Abstract This paper described a method for quantification of ulifloxacin, the active metabolite of prulifloxacin in human plasma by capillary zone electrophoresis using lomefloxacin as the internal standard. The separation was carried out at 25 °C in a 60.2 cm × 75 μm fused-silica capillary with an applied voltage of 20 kV using 200 mM borate buffer (pH 10.5). The detection wavelength was 275 nm. Clean-up and preconcentration of the samples were developed by 96-well format solid-phase extraction. 0.25 ml of plasma sample and 0.25 ml of IS were loaded onto the preconditioned wells, and the wells were washed using 1 ml of 20% methanol in acid water (1% phosphoric acid), and the analytes were eluted using 1 ml of 95/5 methanol/ammonia water. The method was suitably validated with respect to stability, specificity, linearity, lower limit of quantitation, accuracy, precision, extraction recovery and robustness. The calibration graph was linear for ulifloxacin from 0.02 to 2 μg/ml. The lower limit of quantification was 0.02 μg/ml. The intra- and inter-day precisions were within 4.0 and 8.2%, respectively. The method developed was successfully applied to the evaluation of clinical pharmacokinetic study of prulifloxacin formulation product after oral administration to healthy volunteers.