Published data suggest that therapeutic drug monitoring of human immunodeficiency virus protease inhibitors would improve the management of antiretroviral therapy. The authors have developed a high-pressure liquid chromatographic assay allowing simultaneous determination of six protease inhibitors (ritonavir, saquinavir, indinavir, nelfinavir, amprenavir, and lopinavir), using carbamazepine as internal standard. Detection was based on a dual wavelength ultraviolet spectrophotometer and can be improved by the use of a photodiode array detector. Monitoring was performed 1 month after initiation of therapy or in instances of therapeutic failure, side effects, suspicion of noncompliance, drug interactions, or malabsorption. Trough concentrations were 0.15 to 13.6 mg/L for ritonavir, 0.06 to 9.7 mg/L for indinavir, 0.03 to 5.5 mg/L for saquinavir, and 0.15 to 4.15 mg/L for nelfinavir. Concentrations below the limit of quantification were observed in 63/438 (14%) of the patients. Target concentrations are not well established, and reported in vitro inhibitory concentrations may be of limited value. The authors therefore chose to compare observed concentrations with mean plasma concentrations reported in clinical trials. Observed saquinavir and indinavir concentrations were often below or close to these target concentrations, particularly when used as a single protease inhibitor. Concentration-controlled studies should now be used to select proper target concentrations for each protease inhibitor, either prescribed alone or in combination.