A rapid and simple HPLC method was developed for the determination of xanthonolol concentration in plasma. The sample preparation utilized liquid-liquid extraction before injection into the HPLC system. Phenazine was used as the internal standard. Separation was obtained using a reversed-phase column under isocratic conditions. The mobile phase consisted of a 65% McIlvaine buffer containing 0.05% triethylamine adjusted to pH 6.4 with phosphoric acid, 18% acetonitrile, and 17% methanol. Xanthonolol was detected at the ultraviolet wavelength 235 nm. The lower limit of quantization was 50 ng/mL. The assay was applied to a pharmacokinetic study in rats. The plasma concentration of xanthonolol versus time data were best fitted to a two-compartment open model with firstorder elimination processes. After the intravenous administration of xanthonolol at three different dosages of 5, 10 and 15 mg/kg, the respective pharmacokinetic parameters, such as apparent volume of distribution, half-life, and clearance, showed no significant difference at three different dosages. Also, the area under the plasma concentration time curves for three dosages increased proportionally with dose. Therefore, the pharmacokinetics of xanthonolol was found to be linear over the dose range studied.