A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed to determine concentrations of posaconazole in human plasma precipitated by acetonitrile including internal standard. Rapid chromatographic separation was achieved in the mobile phase composition of acetonitrile, water and formic acid (v/v/v, 55:45:0.1) with a flow rate of 0.25 ml/min. Posaconazole-d4 was used as internal standard. Detection was undertaken with cation electrospray tandem mass spectrometry on a Sciex/API3000. The method was accurate, specific and sensitive for the analysis of posaconazole in human plasma in the concentration range of 2–1000 ng/ml. The inter- and intra-batch accuracy was within ± 10% and the lower limit of quantification was 2 ng/ml. The method facilitated a clinical pharmacokinetic study after oral administration of a single-dose of posaconazole suspension in the fasted state and with a high-fat meal in a two-period crossover design. C_max (maximum concentration) and AUC (area under serum drug concentration) were significantly increased, and T_max (time to maximum plasma concentration) was delayed under fed condition, which suggested that simultaneous administration of posaconazole with food may help to achieve higher plasma concentrations and result in better antifungal efficacy.