A simple and rapid high-performance liquid chromatographic method with UV detection is developed and validated to determine the concentration of voriconazole in rat and beagle dog plasma. After protein precipitation using acetonitrile, the supernatant solution is chromatographed on a Diamonsil C 18 column (250 x 4.6-mm i.d., 5 μm). The mobile phase used is a combination of acetonitrile-water-acetic acid (55:45:0.25, v/v/v) with a pH of 4.0. Detection is achieved by a UV detector monitored at a wavelength of 256 nm. The matrix calibration curves are obtained both in the concentration range of 0.10-50.0 μg/mL in rat and beagle dog plasma, with the lower limit of quantitation of 0.10 μg/mL. The intra- and inter-assay precisions in terms of % relative standard deviation are lower than 8.6% and 6.0% in rat and beagle dog plasma, respectively. The accuracy in terms of % relative error ranged from -0.5% to 8.0% and -0.5% to 6.0% in rat and beagle dog plasma, respectively. This validated method is successfully applied to determine the concentration of voriconazole in plasma after intravenous administration of 36 mg/kg voriconazole to rats and 10 mg/kg voriconazole to beagle dogs, respectively.