The aim of this study was to verify the validity of a new self-reported questionnaire designed to assess nonadherence to antiretroviral medication among patients with HIV. Two hundred fifty-six patients from four clinics participated in a prospective longitudinal study. The questionnaire was designed to measure if patients with HIV were taking less than the total number of antiretroviral pills prescribed by their physician. Change in viral load was used as the criterion for validity analyses. Self-reported adherence, viral load and CD4 cell count were assessed at T0 (baseline), T3 (3-month), and T6 (6-month). The findings indicated that the questionnaire had adequate validity (sensitivity, 71%; specificity, 72%; correct classification, 72%; odd ratio, 6.15). These best values were obtained when the analyses excluded individuals with an unstable viral load and a CD4 cell count of less than 200 copies per milliliter over the 6-month follow-up period. This study has shown that this questionnaire has satisfactory psychometric qualities to assess nonadherence to antiretroviral medication among patients with HIV. The questionnaire is brief, simple, and can be used in both clinical or research settings regardless of the patients' antiretroviral regimens.