Methods for HPLC analysis of protease inhibitors (PIs) in human biological matrices were reviewed. Assays have been developed for analysis of single PIs or for simultaneous measurement of multiple PIs in plasma-serum, saliva, cerebrospinal fluid and semen. Liquid-liquid extraction was most often applied for sample pretreatment, but solid-phase extraction and protein precipitation were used as well. Reversed-phase or ion-pair chromatography have been used to separate PIs. Detection of PIs should be sensitive enough for quantitation of plasma concentrations below trough levels of single PIs, or below proposed therapeutic thresholds for PIs. The large majority of assays employs UV detection. As the potential for interferences is large, the selectivity of every method should be evaluated properly. The available high-performance liquid chromatography (HPLC) methods have been applied in clinical pharmacokinetic studies and for therapeutic drug monitoring of PIs. Participation in an interlaboratory quality control program is recommended for every laboratory engaged in the bioanalysis of PIs.