Past research documents multiple factors associated with girls’ susceptibility to human immunodeficiency virus transmission; yet a literature review found no systematic approach to measure vulnerability. This study characterized, developed and tested a set of indicators to measure girls’ vulnerability, resulting in the vulnerable girls index (VGI). A quasi- experimental, separate-sample pre-/post-test design was used to test the index. Adolescent girls were randomly drawn for the pre-test (2277 respondents) and post-test (1418 respondents) from 16 purposively selected communities in Botswana, Malawi and Mozambique. The higher the VGI score—or the more vulnerable the girl—the more likely she was to report premarital sexual experience across the three countries and the more likely she was to report low agency to insist upon condom use in Botswana and Mozambique. The VGI can be used to assess girls’ vulnerability levels across time and space for policy and programme planning purposes, and as part of programme evaluations.