Abstract: OBJECTIVE To establish a LC-MS/MS method for simultaneous determination of the concentration of six antifungal drugs(fluconazole, voriconazole, posaconazole, itraconazole, hydroxy itraconazole, caspofungin) in human plasma. METHODS The plasma samples(50 μL) were precipitated with methanol containing isotope internal standards. The chromatographic separation was performed on a Kinetex C₁₈ column(3 mm×100 mm, 2.6 μm) using a mobile phase of 0.1% formic acid-water(containing 5 mmol·L^-1 ammonium acetate solution) and 0.1% formic acid-methanol, at a flow rate of 0.6 mL·min^-1. The injection volume was 5 μL and the analysis time was 5 min. The detection of the analytes was performed by electrospray ionization in positive mode by multiple reaction monitoring with the transition of m/z 307.0→220.2(fluconazole), m/z 350.1→224.1(voriconazole), m/z 701.4→683.2(posaconazole), m/z 705.2→392.2(itraconazole), m/z 721.5→408.3(hydroxy itraconazole), m/z 547.4→131.1(caspofungin). RESULTS The linear concentration ranges of calibration curves for six antifungal drugs were 0.1-20 μg·mL^-1(r ≥ 0.995 0). The accuracy ranged from 90.35% to 114.94%. The RSD of instruments precision were less than 15%(20% at the lower limit of quantification). The recovery ranged from 71.99% to 90.38% and the matrix effect was between 96.78% and 134.17%. The stability met the acceptance criteria for bioanalytical method validation. The plasma concentrations of antifungal drugs in 25 patients with invasive fungal infection were determined by this method. Tough concentration of voriconazole ranged from 0.70 to 12.94 μg·mL^-1, and fluconazole ranged from 4.57 to 12.64 μg·mL^-1. Significant variance was found among patients. CONCLUSION This method is simple, accurate, sensitive and has a good reproducibility. It is suitable for clinical therapeutic drug monitoring.