Ketoconazole is an imidazole broad-spectrum antifungal agent used for systemic and local infections. The pharmacokinetic information for ketoconazole is relatively limited. The validated method for determining ketoconazole in plasma rabbit is not yet reported. The HPLC-UV method is simple, rapid, cost-effective, sensitive, and only requires a small blood sampling. The chromatographic system consisted of a reversed-phase C18 column (250 x 4.6 mm, 5 µm) at a flow rate of 1 ml/min and detection wavelength of 240 nm, and the retention time of about 5 minutes for ketoconazole and 11 min for itraconazole as internal standard. The calibration curve presented linearity in the 0.05-8 µg/ml with R2=0.9969, which indicated good linearity. The method's precision was obtained by intra-day and inter-day repeatability studies, and accuracy was examined by %diff. The result is <15% for LLOQ and <20% for low, medium, and high levels. Plasma stability under storage in a freezer (-200C) for three days was studied. The validated analytical method was successfully applied to determine the pharmacokinetics parameter of KTZ after a single oral administration.