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To assess the validity of self-reported antiretroviral therapy use (ART) using population-based cohort data.Self-reported ART use and nonuse was compared with a validated laboratory assay in 557 HIV-positive participants in the Rakai Community Cohort Study surveyed between September and December 2011 in Rakai, Uganda. The study population included participants from seven communities, including one fishing community with high HIV prevalence (∼41%). ART use was assayed using liquid chromatography–tandem mass spectrometry, which detects 20 antiretroviral drugs. HIV viral load measurements were also obtained. Individuals with at least two antiretroviral drugs detected were considered to be using ART.One hundred and fifty-three (27%) participants self-reported ART use of whom 148 (97%) had at least two antiretroviral drugs detected. There were at least two antiretroviral drugs detected in 11% (n = 44/404) of individuals with no self-reported ART use. Overall, the specificity of self-reported ART use was 99% (95% CI 97–100%) and the sensitivity was 77% (70–83%). Positive and negative predictive values were 97% (95% CI 93–99%) and 89% (95% CI 86–92%), respectively. Nondisclosure of ART use was significantly more common in younger persons (<30 years) and among those in trading occupations.Self-reported ART use has high specificity and moderate sensitivity providing reasonable, but conservative estimates of population-based ART use. There is more under-reporting of ART use among younger persons and traders suggesting a need for more research on barriers to self-reporting of ART use in these sub-groups.