A rapid high-performance liquid chromatographic (HPLC) method for the determination of levofloxacin in human plasma and urine has been validated. A single-step liquid-liquid extraction procedure was used to isolate levofloxacin from the biological matrix prior to quantitative analysis. The compound was separated on an Inertsil C₁₈ reversed-phase HPLC column and quantified by measuring the UV absorbance at 330 nm. The stereospecificity was achieved in the ligand-exchange mode by incorporating chiral reagents directly into the HPLC mobile phase. Ciprofloxacin was used as the internal standard. The method was linear from 0.08 to 5.18 μg ml⁻¹ of levofloxacin in plasma and from 23 to 1464 μg ml⁻¹ in urine. The overall utility of the method is reflected in its high sample throughput and easy adaptability to robotic automation, thus making the procedure suitable for pharmacological and pharmacokinetic studies of levofloxacin.