The purpose of this study was to validate a reversed-phase high-performance liquid chromatographic (HPLC), tandem mass spectrometry (MS/MS) assay for the determination of telaprevir and its R-diastereomer (VRT-127394) in acidified and nonacidified human plasma. The chromatographic baseline separation of telaprevir and telaprevir-R was performed on a Waters XBridgeTM BEH Shield C18, 2.1 × 75 mm column with a 2.5 µm particle size, under isocratic conditions consisting of a mobile phase of 50:45:5 water–acetonitrile–isopropanol with 1% ammonia at 0.2 mL/min. This method utilized a stable isotope internal standard with 11 deuterium atoms on the structure of the telaprevir molecule (telaprevir-d11). An internal standard for the telaprevir-R (telaprevir-R-d11) was also prepared by incubating telaprevir-d11 in basic solution, which facilitated isomer inter-conversion. The detection and quantitation of telaprevir, telaprevir-R, telaprevir-IS and telaprevir-R-IS was achieved by positive ion electrospray (ESI+) MS/MS detection. The assay quantifiable limit was 5.0 ng/mL when 0.100 mL of acidified human plasma was extracted. Accuracy and precision were validated over the calibration range of 5.0–5000 ng/mL. It was demonstrated using patient samples that, contrary to previous recommendations, quantitation of telaprevir does not require acidified plasma. Copyright © 2014 John Wiley & Sons, Ltd.