A bioanalytical method for the determination of most commonly prescribed protease inhibitors (atazanavir, darunavir, lopinavir and ritonavir) and non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine) was developed and validated according to FDA guidelines. In brief, dried blood spots were punched out of a collection paper with a 0.25 in. diameter punch. The analytes were extracted from the punched-out disc using a mixture of acetonitrile, methanol and 0.2M zinc sulphate in water (1:1:2, v/v/v) containing the internal standards dibenzepine, 13C6-efavirenz and D5-saquinavir. 20 microL of the extract was injected onto the reversed-phase C18 column (150 mm x 2.0 mm) for separation from endogenous compounds and the analytes were quantified using a triple quadrupole mass spectrometer. The analytical run time was only 10 min. Validated concentration ranges covered the ranges encountered in routine clinical practice. The assay was linear over the concentration ranges tested (0.1-20 mg/L for atazanavir, lopinavir, nevirapine and efavirenz and 0.05-10 mg/L for darunavir and ritonavir). Accuracies and inter- and intra-run precisions at all levels ranged from 96.2 to 113.9% and 3.1 to 13.3%, respectively. Analytes in dried blood spots were stable for at least 7 days at 30 degrees C. The method enabled patient-friendly sample collection, easy and cheap sample shipment and non-hospital based sampling for therapeutic drug monitoring and pharmacokinetic studies.