The concentration of darunavir (DRV) in peripheral blood mononuclear cells (PBMCs) was reported to affect the clinical symptoms of patients and the development of drug-resistant viruses. We developed a simple and highly sensitive method to quantify the concentration of DRV in human PBMCs using high-performance liquid chromatography with fluorescence detection. PBMC samples were collected using commercially available tubes for density-gradient centrifugation. To disrupt the cells, we used a heating fragmentation method. To compensate for the disadvantages of the heating fragmentation method, liquid-liquid partitioning was used to destroy the unbroken cells by the degeneration of proteins using an organic solvent. As an analytical column, we used an ODS column. The mobile phase consisted of 20 mmol/L potassium phosphate buffer (pH 4.3)/acetonitrile (57/43, v/v) and was pumped at 1.0 mL/min. The lower limit of quantification was 5 ng/10(6) cells. Good linearity was obtained with 5-100 ng/10(6) cells. The intra- and inter-assay precision and accuracy were <15%. Because our method makes possible the measurement of DRV concentration in PBMCs at medical facilities or laboratories without tandem mass spectrometry systems, we believe that it will contribute to clinical studies and will improve the medical treatment of human immunodeficiency virus infections with DRV.