We have developed and validated a high-performance liquid chromatography method coupled with a mass detector to quantify itraconazole, voriconazole, and posaconazole using quinoxaline as the internal standard. The method involves protein precipitation with acetonitrile. Mean accuracy (percent deviation from the true value) and precision (relative standard deviation percentage) were less than 15%. Mean recovery was more than 80% for all drugs quantified. The lower limit of quantification was 0.031 microg/ml for itraconazole and posaconazole and 0.039 microg/ml for voriconazole. The calibration range tested was from 0.031 to 8 microg/ml for itraconazole and posaconazole and from 0.039 to 10 microg/ml for voriconazole.