Virginia Johnson was born Mary Virginia Eshelman in Springfield, Missouri, the daughter of Edna (née Evans) and Hershel "Harry" Eshelman, a farmer. Her paternal grandparents were members of the LDS Church, and her father had Hessian ancestry. When she was five, her family moved to Palo Alto, California, where her father worked as a groundskeeper for a hospital. The family later returned to Missouri and farming. Virginia enrolled at her hometown's Drury College at age 16, but dropped out and spent four years working in the Missouri state insurance office. She eventually returned to school, studying at the University of Missouri and the Kansas City Conservatory of Music, and during World War II began a music career as a band singer. She sang country music for radio station KWTO in Springfield, where she adopted the stage name Virginia Gibson.
Johnson moved to St. Louis, Missouri, where she became a business writer for the St. Louis Daily Record. Eschewing a singing career, Johnson enrolled at Washington University in St. Louis, intending to earn a degree in sociology but never attaining one.
Johnson met William H. Masters in 1957 when he hired her as a research assistant at the Department of Obstetrics and Gynecology at Washington University in St. Louis. Masters trained her in medical terminology, therapy, and research during the years she worked as his assistant. Together they developed polygraph-like instruments that were designed to measure sexual arousal in humans. Using these tools, Masters and Johnson observed and measured about 700 men and women who agreed to engage in sexual activity with other participants or masturbate in Masters' laboratory. By observing these subjects, Johnson helped Masters identify the four stages of sexual response. This came to be known as the human sexual response cycle. The cycle consists of the excitement phase, plateau phase, orgasmic phase, and resolution phase. In 1964, Masters and Johnson established their own independent nonprofit research institution in St. Louis called the Reproductive Biology Research Foundation. The center was renamed the Masters and Johnson Institute in 1978.

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