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Objective methods of measuring antiretroviral adherence are limited. We assessed the relationship between tenofovir disoproxil fumarate (TDF) hair concentrations, self-reported adherence, and virological outcomes in HIV-infected adolescents in Harare, Zimbabwe. HIV-infected adolescents on atazanavir/ritonavir-based second-line treatment for >6 months with viral load (VL) ≥1,000 copies/mL were randomized to either modified directly administered antiretroviral therapy (mDAART) or standard of care. Hair and VL samples were collected at baseline and after 90 days. Treatment outcome was defined as TDF concentrations in hair. Virological suppression was defined as VL <1,000 copies/mL. Thirty-four adolescents had TDF concentrations measured at baseline and follow-up. Mean (median); range age was 16 (16); 13–18 years and 53% were females. Nineteen (56%) were randomized to mDAART. Mean (SD); range TDF concentrations were 0.03 (0.04); 0–0.17 ng/mg hair and 0.06 (0.06); 0–0.3 ng/mg hair at baseline and follow-up, respectively. Higher TDF concentrations were associated with decreased VL [regression coefficient (RC) 0.8; 95% confidence interval (CI) 0.7–1.0; p = .008] and mDAART (RC 0.5; 95% CI 0.3–1.0; p = .04), but were not associated with self-reported adherence and virological suppression (VL <1,000 copies/mL). Higher TDF hair concentrations were observed with virological decrease and an adherence intervention. Hair antiretroviral concentrations could be useful in triggering adherence interventions among adolescents with second-line virological failure. [ABSTRACT FROM AUTHOR]