A simple, high‐throughput and specific high‐performance liquid chromatography–tandem mass spectrometry method has been developed and validated according to the FDA guidelines for quantification of ulifloxacin in rat and rabbit plasma. The analyte was separated on a Peerless basic C18 column (33 × 4.6 mm, 3 µm) with an isocratic mobile phase of methanol–water containing formic acid (0.5%, v/v; 9:1, v/v) at a flow rate of 0.5 mL/min. The MS/MS detection was carried out by monitoring the fragmentation of m/z 350.500 → 248.500 for ulifloxacin and m/z 332.400 → 231.400 for ciprofloxacin (internal standard; IS) on a triple quadrupole mass spectrometer. The response to ulifloxacin was linear over the range 0.010–2.500 µg/mL in both plasma. The limit of detection and lower limit of quantification of ulifloxacin were determined in both species to be 0.0025 and 0.010 µg/mL, respectively. The method was successfully applied to quantitatively assess the toxicokinetics of ulifloxacin in rat and rabbit following a single 400 mg/kg (in rat) and 200 mg/kg (in rabbit) oral dose of the prulifloxacin. Copyright © 2010 John Wiley & Sons, Ltd.