Abstract: Objective To develop a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of voriconazole in human plasma and its bioequivalence. Methods 48 healthy male volunteers received a single dose of 200 mg voriconazole tablets in a two period (with two preparations) and randomized crossover bioequivalence study. Their plasma voriconazole was determined using HPLC-MS/MS. The pharmacokinetic parameters and bioequivalence of the two preparations were calculated with WinNonlin?6.1. Results The calibration curve of voriconazole ranged from 1 to 5 000 ng/mL. The HPLC-MS/MS method had less than 11% intra- and inter-day relative standard deviation (RSD), with 100.00% to 109.73% accuracies. The RSD of the matrix effect of voriconazole adjusted with internal standard was less than 15%. The extract recoveries exceeded 50% with good stability. The 90% confidence intervals for the peak concentration (Cmax) and the area under the curve (AUC0-t and AUC0-∞) of voriconazole fell into the bioequivalence range of 80.00%-125.00%. There was no significant difference in peak time (Tmax) between the two preparations. Conclusion HPLC-MS/MS can be used for determination of voriconazole in human plasma. The two tested preparations of voriconazole are bioequivalent.