Posaconazole is a new broad-spectrum antifungal agent that is currently only available as an oral suspension and shows high intra- and inter-individual differences in oral bioavailibility. Pre-existing methods for the determination of the substance involve the use of internal standards or require a quite complicated and time-consuming sample pre-treatment. Our HPLC method is fast and fully-automated and there is no need for any manual sample pre-treatment. On-line transfer of posaconazole from the extraction column was followed by chromatographic separation on a C18 column and fluorescence detection (λex: 261 nm, λem: 357 nm). Retention time of posaconazole was about 11.7 min, the lower limit of quantification was found to be 0.1 mg/l. A linear calibration curve was obtained over the concentration range of 0.1-5 mg/l using a 50 μl sample (r2 = 0.999). The relative standard deviations of intra-day variations ranged from 2.3% to 9.4%, intra-day accuracy from 88.8% to 114.8%. © 2009 Elsevier B.V. All rights reserved.