The development and validation of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of itraconazole and its metabolite, hydroxyitraconazole, in human plasma is described. The method involved liquid-phase extraction of itraconazole and hydroxyitraconazole using a hexane-dichloromethane (70:30) mixture, after addition of loratidine as an internal standard (IS). Separation was achieved with a reversed-phase C18 column (250 mm x 4.6 mm) employing fluorescence detection (excitation: 264 nm, emission: 380 nm). The mobile phase consisted of [0.01% triethylamine solution adjusted to pH 2.8 with orthophosphoric acid-acetonitrile (46:54)]-isopropanol (90:10, v/v) at a flow rate of 1.0 ml/min. For both the drug and metabolite, the standard curve was linear from 5.0 to 500 ng/ml with goodness of fit (r2) greater than 0.98 observed with four precision and accuracy batches during validation. An observed recovery was more than 70% for drug, metabolite and internal standard. The applicability of this method to pharmacokinetic studies was established after successful application during 35 subjects bioavailibity study. The method was found to be precise, accurate and specific during the study.