Introduction: Now that HIV infection has become a chronic disease, optimizing health status is an important goal of care for HIV-infected patients. Testosterone insufficiency (TI) can compromise health status, but little is known about the prevalence of TI and possible related factors in HIV-infected women. Aim: To investigate the prevalence of TI among HIV-infected women attending our HIV outpatient clinic, and to study the relationship between TI and sexual function, fatigue, health status, and depression. Methods: 56 HIV-infected women aged ≥18 years who attended the HIV outpatient clinic of the Amsterdam University Medical Center, The Netherlands, were included. Blood samples were taken for endocrinologic testing and patients filled out 6 validated questionnaires measuring sexual function, fatigue, health, and depression. Main Outcome Measure: TI, the Female Sexual Function Index, the Female Sexual Distress Scale-Revised, the Multidimensional Fatigue Inventory, the Medical Outcomes Studies Short Form 36-item health survey, and the Beck Depression Inventory were assessed. Results: A relatively high prevalence of TI, 37%, was found. Plasma viral load and CD4 cell count did not differ between women with or without TI. Clinical fatigue, physical fatigue, and impaired cognitive function were significantly more prevalent in women with TI. Women with TI also tended to report decreased sexual desire, reduced physical activity, increased mental fatigue, reduced physical function, increased health distress, and clinical depression. Conclusion: We recommend that in all HIV-positive women with complaints typical for TI, testosterone is measured, and that in women with TI, testosterone replacement be considered as a treatment option. However, given that complaints are also prevalent in HIV-positive women without TI, the approach to women with these complaints should include sexual and psychological evaluation. Laan ETM, Prins JM, van Lunsen RHW, et al. Testosterone Insufficiency in Human Immunodeficiency Virus–Infected Women: A Cross-Sectional Study. Sex Med 2019;7:72–79.