A high-performance liquid chromatographic method was developed for the determination of a new derivative of docetaxel, felotaxel, in rat plasma and human plasma and urine. The separation of felotaxel was performed on a Dikma C18 column with 0.2% formic acid and acetonitrile (50:50) as the mobile phase. The flow rate was 0.8 mL/min and the column effluent was monitored by an ultraviolet detector set at 275 nm. The method was validated and found to be linear in the range of 5-1,000 ng/mL. The lower limit of quantification was 5 ng/mL based on 100 µL of plasma. The variations for intra-day and inter-day precision were less than 6.9%, and the accuracy values were between 87.3 and 107.4%. The extraction recoveries were more than 80.5%. These data confirm that the developed method has satisfactory sensitivity, accuracy and precision for the quantification of felotaxel in rat plasma and in human plasma and urine. The method was successfully applied to a pharmacokinetics study of felotaxel after intravenous doses of 5 mg/kg in rats.