A simple, rapid and selective high-performance liquid chromatographic (HPLC) method for the determination of sparfloxacin in human plasma has been developed and evaluated. Plasma protein was precipitated with acetonitrile. The drug and the internal standard (furosemide) were eluted from a Nova Pak C18cartridge column at 50°C with a mobile phase consisting of 5% acetic acid:acetonitrile:methanol (70:15:15% v/v). The column eluent was monitored at 364 nm. Each analysis required no longer than 5 min. Quantification was achieved by the measurement of the peak-area ratio of the analyte to the internal standard and the limit of quantification for sparfloxacin in plasma is 25 ng/ml. The intraday coefficient of variation (CV) ranged from 1.71% to 6.1%, and interday CV from 2.60% to 4.28% at four different concentrations. The absolute recoveries ranged from 98.6% to 104%, and the relative recoveries from 93.6% to 116.4% at four different concentrations. Preliminary stability tests showed that sparfloxacin is stable for at least 4-weeks in human plasma after freezing. The method is applied for the determination of the pharmacokinetic parameters of sparfloxacin after oral administration to 3 rats.