Gladys E. Hamlin was born in Sioux Falls, South Dakota and received her Bachelor of Philosophy (Ph.B.) degree at the University of Chicago in 1926. After teaching in high schools in South Dakota and Iowa, she entered Columbia University where she pursued her Master of Arts degree in art history (1931–1937). Following her graduation from Columbia, she accepted a teaching position at Duke University and taught art history from 1938 to 1939.

In 1944, Hamlin joined the staff of the American Council of Learned Societies where she assisted with gathering information on the looting and damage of art in Europe. In 1945, she transferred to the Roberts Commission, serving as William L.M. Burke’s assistant on the continuation of the ACLS research project until 1946. During this time, she published three articles in the College Art Journal, reporting on the events: “European Art Collections and the War” (March 1945); “European Art Collections and the War: II” (January 1946); and “German Paintings in the National Gallery” (March 1946).

Following World War II, Hamlin worked as a docent at the National Gallery of Art in Washington, D.C. from 1946 to 1949. At that time, she accepted a position as Associate Professor at Iowa State University and she remained at the university as Professor of Art History until her retirement in 1973.

The Gladys E. Hamlin papers, containing her ACLS and Roberts Commission papers, reside in the Iowa State University Archives at Ames, Iowa.