OBJECTIVES: This study aims to present a strategy to optimize a liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for voriconazole and voriconazole-N-oxide with an stable isotopically labelled internal standard. METHODS: Protein precipitation was used as extraction method and detection was carried out with LC-MS/MS using 13C2-2H3-voriconazole as internal standard. RESULTS: Voriconazole and voriconazole-N-oxide concentrations can be detected with good accuracy and reproducibility within the therapeutic range (ref. 1-6 mg/L). Accuracy ranged from -3.0%-4.2% for voriconazole and -1.0%-3.8% for voriconazole-N-oxide and overall coefficients of variation (CV) ranged from 2.9%-7.5% for voriconazole and 4.0%-10.8% for voriconazole-N-oxide. However, voriconazole-N-oxide is relatively unstable at room temperature (Low QC sample: -81.1% after 120 hours), therefore samples should be cooled (2-8°C) after sampling to detect reliable voriconazole-N-oxide concentrations. CONCLUSIONS: An accurate and simple assay for the analysis of voriconazole and voriconazole-N-oxide to enable therapeutic drug monitoring was developed and validated for all critical parameters.