Background: Voriconazole (VRZ) is a triazole antifungal used for treatment of invasive fungal infection, which is a life-threatening condition. Therapeutic drug monitoring is recommended for identifying the optimal dose in patients who have hepatic/renal impairment or reduced function of the CYP2C19 metabolizing enzyme. Methods: One hundred microliters of sample plasma was extracted by protein precipitated with 200 mu l of acetonitrile containing fluconazole as internal standard (IS). After vortexing and centrifugation, supernatant was dried and reconstituted with 100 l of mobile phase (ACN: 0.1% formic acid in 10 mM Ammonium acetate) (50:50 v/v) before injected. The column was C18, 2.7 m, 3.0 x 50 mm at flow rate of 0.5 ml/min with retention time of 0.5 and 0.75 min for VRZ and IS, respectively. The tandem mass spectrometer was set in multiple reactions monitoring (MRM) mode with the following transition; VRZ m/z 350.10 ->281.10 and 307.20 ->220.20 (IS). Results: The accuracy and precision inter- and intra-day were less than 9%, over the range 0.05-10 mu g/ml. The linearity was consistent (r(2) = 0.9987) and recovery was more than 85.0% for both analyses. Conclusion: This method is applicable for routine monitoring of patients' VRZ plasma level with fast and accurate runtime to assess CYP2C19 genotype. (C) 2016 Wiley Periodicals, Inc.