Accurate, routine measurement of recent illicit substance use is challenging. The Johns Hopkins Human Immunodeficiency Virus Clinical Cohort (Baltimore, Maryland) collects 2 imperfect but routine measurements of recent substance use: medical record review and self-interview. We used Bayesian latent class modeling to estimate sensitivity and specificity of each measurement as well as prevalence of substance use among 2,064 patients engaged in care during 2007-2015. Sensitivity of medical record review was higher than sensitivity of self-interview for cocaine and heroin use; posterior estimates ranged from 44% to 76% for cocaine use and from 39% to 67% for heroin use, depending on model assumptions and priors. In contrast, sensitivity of self-interview was higher than sensitivity of medical record review for any alcohol use, hazardous alcohol use, and cigarette smoking. Posterior estimates of sensitivity of self-interview were generally above 80%, 85%, and 87% for each substance, respectively. Specificity was high for all measurements. From one model, we estimated prevalence of substance use in the cohort to be 12.5% for cocaine, 9.3% for heroin, 48.5% for alcohol, 21.4% for hazardous alcohol, and 55.4% for cigarettes. Prevalence estimates from other models were generally comparable. Measurement error of substance use is nontrivial and should be accounted for in subsequent analyses.