Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC–MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05 ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid–liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10 μL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1 × 50 mm, 4 μm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748 → 648 for asunaprevir and m/z 757 → 649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50 ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.