OBJECTIVES: To evaluate the psychometric properties of the Functional Assessment of Human Immunodeficiency Virus Infection (FAHI) questionnaire, a 47-item disease-specific instrument evaluating Health-Related Quality of Life (HRQL) in Human Immunodeficiency Virus (HIV) patients, and show its usefulness for the evaluation of new antiretroviral therapy. METHODS: Treatment-experienced HIV patients included in two independent clinical programmes completed the self-administered FAHI questionnaire at Baseline and after 24 weeks of treatment. The FAHI questionnaire includes five scales (physical well-being, emotional well-being, functional and global well-being, social well-being, cognitive functioning) and a total score, with higher scores indicating better HRQL. Psychometric properties of the FAHI questionnaire were assessed independently in both trial populations (N=565, 1096). A range of minimal important differences (MID) was provided using anchor-based and distribution-based methods. The link between HRQL and biological endpoints was explored by regression analysis. RESULTS: Internal consistency reliability was good, with Cronbach’s alphas ranging from 0.72 to 0.94. Most items met both convergent and discriminant validity criteria, demonstrating good construct validity of the scores. Clinical validity was demonstrated by better FAHI scores, indicating better HRQL for patients in earlier HIV stages. Changes in scores were significantly linked to the change in EQ-5D score, demonstrating their responsiveness. MID ranged from 3.2 to 14 for the FAHI Total score. Regression analyses between the FAHI Total score and CD4 cell count and viral load showed a poor relationships between HRQL and biological parameters (r-square<3%). CONCLUSIONS: The FAHI questionnaire demonstrated robust psychometric properties in two independent clinical trial populations. The assessment of HRQL enabled the detection of changes in patients’ health status not revealed by traditional clinical parameters of efficacy.