During the 1870s, Upjohn's family lived with her grandfather, a well-known architect named Richard Upjohn who had retired to a scenic home in Garrison, New York. Richard Upjohn's biographer says when she was about five she would accompany him as he sketched and painted. She questioned him about his color choices and learned that artists often chose colors different from the ones present in the subjects they painted. He also showed her engravings of famous paintings, explaining what made them great and where they fell short in his view. Her family was living in Fond du Lac, Wisconsin when she graduated from high school in 1887. A few years later, the family moved to New York, where, in the early 1890s, she took classes at the Cooper Union Woman's Art School. She began her foreign travels in 1893 and during the next few years studied art in Munich, Florence, and Paris. In 1902, she took an illustration class at the National Academy of Design and the following year won the Academy's Suydam silver medal for her work. Between 1909 and 1912, she studied and traveled in Europe's other major cities. In 1922, Upjohn told a reporter that she had studied art "in many places, usually for a few months at a time and disconnectedly, but what counted most was the work she did in Paris under Castelucho and Lucien Simon.” Born in Barcelona, Castelucho's birth name was Claudi Catelucho Diana, but he went by his surname alone. In Paris during the early years of the twentieth century, he and Simon both trained private students and both taught at two mondernist alternatives to the École des Beaux Arts: the Académie Colarossi and the Académie de la Grande Chaumière. Upjohn did not say whether she took private lessons, classes, or both.

In 1890, at the age of twenty-one, Upjohn completed a painting of angels for St. Paul's Episcopal Cathedral in Fond du Lac, Wisconsin. Her uncle Richard M. Upjohn had designed the building and her father was currently its rector (having succeeded John Henry Hobart Brown his brother-in-law). Reporting on the installation of the painting, a local journalist said the angels were "finely executed." The following year, she completed another painting for the cathedral. Measuring eight by fourteen feet, it depicted adoring saints and was placed in the cancel. A local reporter said it reflected "great credit upon the artist." In 1896, Upjohn had a portrait accepted for display in the autumn exhibition at the National Academy of Design. Called an "uncompromising portrait of a lady" by a local critic, the painting was the first of her formal portraits to gain recognition. A decade later, she told a reporter she was particularly proud to be included in this show. During travels to paint and study art in Europe in the 1890s, Upjohn participated in exhibitions held in Baden Baden, Paris, and Munich. In 1901, she showed paintings of children in a solo exhibition held at the gallery of the Kilohana Art League in Honolulu, Hawaii. Reviewing the show, a local critic said "One can be sanely and intelligently enthusiastic over Miss Anna M. Upjohn's child pictures. Anything more ideally perfect in this line than her "Children" and "A Child" is hard to find. Her work is unusually bold. Upjohn traveled to paint and study again during the years between 1900 and 1914. In 1910, she made a characteristic painting of a cheerful boy carrying a fishing pole (see Image No. 1, above).

She was living in Brooklyn in 1912 when she executed a commission from Cornell alumni for a portrait of her uncle, Charles Babcock, a professor of architecture (emeritus) at the university. At this time, she became a member of the MacDowell Club of New York and participated one of the club's exhibitions in 1913. Reviewing this show, a critic for the New York Times singled out a painting of Scandinavian children for its good characterization and "freshness and force of color". During the next few years, Upjohn contributed frequently to MacDowell club exhibitions. In 1914, the prestigious International Studio art magazine reproduced two paintings from a then-current exhibition and its critic called her "one of the best contributors lately at the MacDowell Club". The following year, a critic for the Brooklyn Daily Eagle said a painting of a child seen in a club exhibition was "one of the strongest and best types of youngsters in paint, which can be imagined." During these pre-war years, she also exhibited at the Grand Central, Knoeller, Macbeth, and Anderson Galleries in New York.

Subjects of commissioned formal portraits in this period include Helen Van Vechten, maker of fine-press books; A. Cameron MacKenzie, president of Elmira College; General George Wood Wingate an early leader of the National Rifle Association and founder of a NY boys' club; Mary Williams, an Ithaca matriarch; and notables connected with Cornell University, including Andrew Dickson White, a historian and one of the founders of the university, Louis Agassiz Fuertes, an ornithologist, illustrator and artist, and the anatomist Charles Rupert Stockard.

When she gave a solo exhibition at the Cornell College of Architecture in 1913, the local paper called her work "captivating" and said a painting of a Portuguese fisher girl gave "true joy" in its "fresh execution". Another solo show, this time at the Arnot Gallery of Elmira College, was said to be a "beautiful exhibit" of portraits and paintings of children. In 1915, a Wisconsin seed company bought a painting she had made of her six-year-old sister at work in a garden. The company made posters of the painting to use as promotional materials.

Upjohn's career changed abruptly at the outbreak of the First World War. Happening to be in Paris when war was declared, she volunteered her services to the local American Episcopal church in its efforts to help the refugees who poured into the city. On her return to New York in 1915, she solicited funds to support relief organizations and sold her drawings and paintings to raise money for them. She returned to France in 1916 and spent most of her time doing relief work there and in Belgium during the rest of the war. When, in 1917, the Paris-based Children's Bureau of the American Red Cross (ARC) asked her to make paintings for a series of health posters, she was reluctant to take time away from her relief work but she made five canvases for them all the same. In 1919, Upjohn visited war-devastated countries to make drawings and paintings for the Junior Red Cross (JRC). The ARC had set up the American Junior Red Cross in 1917 with three main objectives: to educate American children about the war and its impact on European children, to obtain donations of clothing and other goods, and to solicit donations for relief work. Upjohn's role was to make works of art depicting European children in a favorable light: cheerful and friendly despite their difficult circumstances. In September 1919, a striking drawing by Upjohn appeared in the first issue of the ARC's Junior Red Cross News with the caption, "This picture of boys and girls of France, with their beloved Tricolor, served as a poster in the Child Welfare Exhibits held in various cities of France a few months ago by the Children’s Bureau of the American Red Cross. It is the work of Miss Anna Milo Upjohn, a New York portrait painter, with the American Red Cross in Europe." See Image No. 2, above.

Upjohn's career reached another turning point in 1921 when she joined the JRC as a staff artist. She was then 53. Her professional career had begun thirty years earlier and would continue another two decades. Her new position gave her financial stability and welcome opportunities to travel. It did not prevent her from continuing to make and exhibit her paintings and drawings. In 1921, the Corcoran Gallery in Washington, D.C. mounted a solo exhibition of drawings she had made in Europe while on assignment for the JRC. Reproductions of the drawings had previously been printed in issues of Junior Red Cross News and, in announcing the exhibition, the journal's editor had restated the organization's objective as giving American children "an intelligent, sympathetic understanding" of the children in war-torn European countries. The show generated considerable publicity in Washington with one critic saying Upjohn had "presented not only the pathetic, but the humorous, and has in many instances given her sketches the homely touch which makes for universal appeal." Subsequently put on tour across the country, the drawings continued to draw critical notice, including a comment that the drawings showed a "homely touch" making for "universal appeal". Early in 1923, Upjohn visited the American West for the JRC to sketch Indian children and later that year the organization sent her on a tour around the world from west to east. After her arrival in Honolulu, a reporter described her child studies as "authoritative" and effective in achieving the JRC's goals of sympathy and understanding. On her return to the United States, the Maryland Institute in Baltimore exhibited a large number of her drawings from East- and Southeast Asia as well as Western, Eastern, and Southern Europe, the collection showing, as one critic said, "not pictures of children suffering, but happy childhood under varying conditions of life".

In 1935, the Cronyn & Lowndes Gallery in Manhattan gave Upjohn an exhibition of her paintings, including both formal portraits and informal child studies. Writing in the New York Times, the critic, Howard Devree, saw "competent, honest work rather on the conventional side". Another critic noted the significance of the formal portraits but said the child studies were the most interesting element in the show.

Upjohn resigned her position at the Junior Red Cross in 1931, but she continued to make drawings for its publications. During the 1930s, the ARC continued to tour exhibitions of her paintings, watercolors, and drawings to public libraries and other exhibition spaces throughout the United States. In Mansfield, Ohio, the local newspaper headlined Upjohn as a "Famous Artist" whose work appeared in ARC posters, JRC literature, and other media.

Exhibitions of Upjohn's paintings and drawings were less frequent from 1940 onward and the last may have been the one held in the public library of Somerset, Pennsylvania, in June 1945.

Upjohn trained as both artist and illustrator. She attracted most critical notice for her portraits and other paintings and invariably put "artist" when asked to give her occupation.She painted in oil on canvas; made gouache, watercolor, and wash drawings; and often worked in charcoal or crayon. She was best known for her formal portraits and informal depictions of children. She also made paintings of religious subjects and some landscapes.

Critics discussed the quality of her portraits without commenting on style. One called an early portrait "uncompromising". Another rated her portrait of John van Benschoten one of the "strongest" in the exhibition where it appeared. (The sitter was probably a brother of Upjohn's close friend, Augusta K. van Benschoten.) Her portrait of a woman named Elizabeth Spencer was said to be "well posed and fluent". Two of her portraits, one showing Andrew Dickson White and the other Louis Agassiz Fuertes were claimed to be "two of the best in the possession of Cornell University".

Her informal portraits of children and other works drew relatively few comments on style. On an exhibition of drawings held in Boston in 1921, a critic said, "The drawings were made, sometimes with charcoal, sometimes with colored crayon. The best, as so often happens, are those produced with most evident haste and spontaneity. Here and there one notes a little of that elaboration that takes the crisp edge off. In general, however, the childish forms are drawn with a taut, nervous line and with brisk indication of planes and attachments." In 1935, Howard Devree of the New York Times commented on the pastel effects she achieved, "despite use of a palette knife, through harmonizing of colors". Another critic noted that Upjohn's informal portraits were usually done outdoors and almost never in her studio.

Among the contributors to the magazine were editor Gene Gauntier, and authors Temple Bailey, Ellis Parker Butler, Rachel Carson, Arthur Guiterman, Patricia Highsmith, Shirley Jackson, Anita Loos, Neysa McMein, Kathleen Norris, Sylvia Schur, John Steinbeck, Willa Cather, Frank Albert Waugh and P. G. Wodehouse. Notable illustrators included Rolf Armstrong, Wladyslaw T. Benda, Elizabeth Shippen Green, Bessie Pease Gutmann, Rico Lebrun, Neysa McMein, Violet Oakley, Herbert Paus, May Wilson Preston, Olive Rush, Arthur Sarnoff and Frederic Dorr Steele.

Spurred on by the success of other mail-order monthlies, two brothers, S.L. and Frederick Thorpe of Cleveland, Ohio started their magazine in 1874. The magazine called The Home was only eight pages in size, produced on cheap paper and the subscription price was fifty cents a year. The content consisted of household articles, fiction by unknown writers and advertisements mostly for mail-order items. A year after Frederick died in 1877, S.L. acquired another Cleveland periodical called Little Ones at Home. Thorpe consolidated both titles under the new title of Home Companion: A Monthly for Young People. According to Thorpe, but not verified officially circulation reached eighty-eight thousand. Thorpe had been studying medicine, and when he started his practice in 1881, he sold the paper to E.B. Harvey and Frank S. Finn. In 1882, after starting a higher-class magazine without advertising called Young Folks' Circle, Harvey & Finn sold The Home Companion to Mast, Crowell, & Kirkpatrick of Springfield, Ohio.

Phineas P. Mast had hired John Crowell of Lexington, Kentucky to launch and manage Farm & Fireside magazine in Springfield, Ohio. P.P. Mast made his money through agricultural equipment and wanted a magazine to promote his wares. Farm & Fireside launched in 1877, and the firm acquired The Home Companion in 1883 after realizing the market for content aimed at women. Crowell then purchased Harvey & Finn's Young Folks' Circle in 1884, which was absorbed into The Home Companion two years later. In November 1886, the name of the periodical was changed to Ladies' Home Companion. Mast's nephew, T.J. Kirkpatrick was the first general editor of the periodical.

During the 1880s, the magazine changed size and length, and the quality of the content was improved by the addition of writers such as Maria Louise Pool, James Otis, and Eben E. Rexford. Now published semi-monthly, an important feature of the magazine was a Practical Housekeeping department, which was created by Eliza R. Parker. Woodcuts were used for illustration and at times the magazine reprinted articles from other magazines. Coverage was given to food, fashion, and serialized fiction. Topics covered included—household budgeting, home building, and furnishing, needlework, health, childcare, and etiquette. By 1889 circulation had reached eighty thousand, and, in 1890, it hit one hundred thousand. It was considered a leader in the field of women's interest magazines.

The cover was created for the first time for the Christmas issue of 1891—covers would not become a regular feature until three or four years later and halftone pictures made from photographs would appear in 1891. It was in 1893 that the price of the magazine was raised to one dollar a year. At the time the Companion competed with the Ladies' Home Journal which was twice as long, only published twelve times a year, and had a much larger circulation. To compete, the Companion went to a monthly publication and cut the price back to fifty cents—at the same time it upped the quality of its articles and writers. Circulation soon rose to 300,000 by 1898—still only half of the Ladies' Home Journal.^[To further the distance—the Companion's name was officially changed to Woman's Home Companion in 1896. According to Frank L. Mott's History of the American Magazine, the editor, presumably Joseph F. Henderson, wrote of the change in the January 1887 edition:

The indiscriminate use and abuse of the term "lady" has robbed it of so much of its meaning that it has been in a measure tabooed by those who deserve the title in its best sense. The noblest ambition of our end-of-the-century femininity is to be a "woman."..."Woman" is an honest Anglo-Saxon word, and has no synonym. The use of "lady" as a synonym for "woman" is vulgar.

During the 1890s in addition to housekeeping tips the magazine also covered subjects such as college education for girls, women in the arts and civil service, travel abroad, women's clubs and health. There was no mention of the war with Spain except for one article on the American Red Cross in the September 1898 issue.

At the turn of the century, the magazine's parent company went through some changes. P.P. Mast died in 1898 and 1901, T. J. Kirkpatrick sold his remaining interests to John Crowell and the main editorial offices were moved to New York (printing was to remain in Springfield). In 1906, Joseph P. Knapp paid $750,000 for controlling interest in the Crowell Publishing Company. During this period the magazine went from twelve pages of advertisements in 1901 to over 75 by 1907.

At the same time, the magazine went through editor and editorial changes as well. Arthur T. Vance became the editor in 1901 and Vance pushed to broaden the scope of the magazine into general interest areas. During this time many magazines were outlets for what was called muckraking journalism—a general movement in journalism from after 1900 until around World War I. Editors and journalists took on investigative reporting to raise public awareness of social issues of the day. Woman's Home Companion was not known as a muckraking magazine, but under Vance's editorial-ship and push towards general interest stories, the magazine featured a crusade against child slavery during 1906-07. Coverage included child-workers in cotton mills, canning factories, tailoring, and sweat shops. The January 1907 issue featured a statement signed by President Theodore Roosevelt entitled, Where I Stand on Child Labor Reform. Under Vance there was coverage of art and music, architecture, books in addition to the regular departments dealing with fashion and the home.

Vance was also interested in short stories and the list of authors who published included Frank H. Spearman, Hamlin Garland, Sarah Orne Jewett, Bret Harte, Robert Grant, Jack London, Eden Philpotts, Morgan Robertson and Rafael Sabatini. Jack London wrote the short story the Apostate, which was published in the September 1906 edition. London also published coverage of his cruise around the South Pacific under the title, Round the World for the Woman's Home Companion . This journey was also the basis for London's book The Cruise of the Snark.