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A convenient and sensitive liquid chromatography–tandem mass spectrometry method was established to simultaneously quantify voriconazole (VRZ) and its metabolite, voriconazole N -oxide (VNO), in human urine. Voriconazole-d3 and voriconazole-d3 N -oxide were used as isotopic internal standards. Samples were processed by protein precipitation and separated using a ZORBAX SB-Aq column (1.8 μm, 2.1 × 50 mm). Mass spectrometry was performed using an API 4000 mass spectrometry by positive electrospray ionization. The flow rate was 0.6 mL/min. Gradient elution was performed with methanol and 0.1% formic acid as the organic and water phase, respectively. The VRZ and VNO calibration curves ranged from 20.0 to 7200 ng/mL in human urine. The specificity, matrix effect, extraction recovery, intra/inter-run precision, accuracy and stability were validated for both VRZ and VNO in human urine. The developed method was used to study urinary excretion after intravenous injection of 4 mg/kg VRZ in healthy Chinese subjects. [ABSTRACT FROM AUTHOR]