A simple, ion-pair high-performance liquid chromatographic method has been developed and validated for the quantitative determination of the HIV protease inhibitor ritonavir in human plasma, cerebrospinal fluid and saliva. Sample pretreatment consisted of precipitation of proteins with acetonitrile prior to high-performance liquid chromatography with ultraviolet detection at 239 nm. The method has been validated over the range of 50 ng/ml to 50 microg/ml with use of 100-microl volumes of sample. The currently described assay has been used successfully for the analysis of ritonavir in plasma, cerebrospinal fluid and saliva in HIV-1 infected patients.