Acquired immunodeficiency syndrome is an infectious disease caused by human immunodeficiency virus (HIV). Pharmacological antiretroviral therapy, along with therapeutic monitoring of drug plasma concentrations, facilitates adequate and effective follow-up in each patient. Our main objective was to develop and validate a high-performance liquid chromatography with ultraviolet detector (HPLC-UV) bioanalytical method for the simultaneous quantification of plasma concentrations of three commonly used antiretrovirals in Colombia: efavirenz, lopinavir, and ritonavir. This method is intended for therapeutic drug monitoring in persons living with HIV locally or in developing countries. The method is selective, specific, accurate, precise, and stable over the concentration range of 250 and 20,000 ng mL -1 for the drugs. It was successfully applied to measure the plasma levels of efavirenz, lopinavir, and ritonavir in HIV patients, which ranged from 251.0 ± 10.4 to 7186.2 ± 10.2, 8188.2 ± 5.9 to 15,312.5 ± 6.6, and 246.1 ± 6.5 to 1189.0 ± 6.4 ng mL-1, respectively. HIV. . (HIV) therapy followup follow up patient highperformance high performance HPLCUV HPLC UV (HPLC-UV Colombia efavirenz lopinavir countries selective specific accurate precise 25 20000 20 000 20,00 1 - drugs patients 2510 251 0 251. 104 10 4 10. 71862 7186 2 7186. 102 10.2 81882 8188 8188. 59 5 9 5. 153125 15 312 15,312. 66 6 6.6 2461 246 246. 65 6. 11890 1189 1189. 64 mL1, mL1 1, mL-1 respectively (HIV 2000 00 20,0 718 818 15312 31 15,312 24 118 mL- 200 20, 71 81 1531 3 15,31 11 7 8 153 15,3 15,