A reliable liquid chromatography-tandem mass spectrometric(LC-MS/MS) method was developed and validated for the simultaneous determination of vancomycin and voriconazole in human plasma. The analytes and internal standard 10-hydroxycarbazepine were separated at a flow rate of 0.9 mL/min using a Zorbax SB-C-18 column (50 mmx4.6 mm, 2.7 mu m). Positive ion electrospray ionization was used to detect vancomycin, voriconazole and intrenal standard(IS) 10-hydroxycarbazepine followed by multiple reaction monitoring(MRM) of the transition at m/z 725.5 -> 144.2, 350.3 -> 281.0 and 253.1 -> 208.0, respectively. The total run time for both vancomycin and voriconazole samples was 5 min; 0.30 mu g/mL was the lower limit of quantification. The precision of intraday and interday was no more than 12.4%. The method was successfully and resoundingly applied in therapeutic drug monitoring of 156 patients treated with vancomycin and voriconazole.