A rapid and sensitive method for the determination of indinavir in mice brain and testis is described and validation data are provided. Indinavir and the internal standard (IS) amprenavir were isolated from homogenized tissue matrices using a mixed-mode solid-phase extraction (SPE) procedure and were then analyzed by reversed-phase liquid chromatography/tandem mass spectrometry (LC-MS/MS). The mass spectrometer in the positive-ion multiple reaction monitoring mode used pairs of ions at m/z of 614.1/421.3 for indinavir and of 506.1/245.3 for IS. The calibration curves were linear over the range 0.0012-0.0390 micromol/kg for brain and 0.39-12.50 micromol/kg for testis. Linearity, repeatability and accuracy were validated. The applicability of the method was demonstrated by assessing indinavir in brain and testis of three mice dosed with intravenous bolus administration of indinavir (16.3 micromol/kg).