Born Rosalyn Sussman in New York City, she graduated in 1941 from Hunter College, where she developed an interest in physics. Not believing that any good graduate school would admit and provide financial support to a woman (and a Jewish woman, at that), Sussman took a job as a secretary to a leading biochemist. However, soon after graduating she received an offer for a teaching assistantship in physics from the University of Illinois. She was the only woman among the department's 400 members, and the first since 1917. She married fellow student Aaron Yalow in 1943, and received her Ph.D. in 1945.
After graduating, Yalow joined the Bronx Veterans Administration Hospital to help set up its radioisotope service. There she collaborated with Solomon Berson to develop RIA, a radioisotope tracing technique that allows the measurement of tiny quantities of various biological substances in the blood. Originally used to study insulin levels in diabetes mellitus, the technique has since been applied to hundreds of other substances – including hormones, vitamins and enzymes – all previously too small to detect. Despite its huge commercial potential, Yalow and Berson refused to patent the method.