Therapeutic drug monitoring (TDM) of voriconazole, itraconazole, and posaconazole is a useful tool for treatment of fungal infections. We validated a simple and reliable LC-MS/MS method using simple protein precipitation for simultaneous determination of voriconazole, itraconazole, and posaconazole. The linearity, accuracy, precision, carryover, and matrix effects were validated. Total sample preparation time was <30 min per batch, and analytical run time was 3.8 min per sample. We also presented clinical experience of TDM at 1183 serum concentrations over one year using this validated assay method. About 77%, 85%, and 96% of measured voriconazole, itraconazole, and posaconazole concentrations were within the therapeutic range, respectively. The number of respective measurements per patient was 1-63, 1-8, and 1-4 for voriconazole, itraconazole, and posaconazole. All three antifungal agents showed large intra-individual variability (2 to 181% CV) and drug-drug interaction with proton pump inhibitors or rifampin. In conclusion, we developed and validated a simple and fast method that was successfully applied in a routine clinical setting.