Objective To establish a rapid, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of acyclovir (the metabolite of valacyclovir hydrochloride) in human plasma. Methods After addition of ganciclovir as internal standard (IS), plasma samples were prepared by one-step protein precipitation using acetonitrile as precipitant, followed by an isocratic elution with 0.1% formic acid 3.5μm) column. Detection was performed on a triple-quadrupole mass spectrometer utilizing electrospray ionization (ESI) interface operating in positive ion and selected reaction monitoring (SRM) mode with the precursor to product ion transitions m/z 226.2→152.1 for acyclovir and m/z 256.2→152.1 for the IS. Results The analytical results demonstrated a good linearity over the ranges from 0.005 to 4μg/mL (r=0.9999) for valacyclovir hydrochloride. The relative standard deviations (RSD) of intra-batch and inter-batch were less than 4.06% and 9.23%, respectively. The limit of detection and lower limit of quantification in human plasma were 2ng/mL and 5ng/mL, respectively. Conclusion The method was simple, sensitive, accurate and reproducible and has been successfully applied to a bioequivalence study of valacyclovir hydrochloride capsules in Chinese healthy male volunteers.