A simple, specific, sensitive, and rapid method has been developed and validated for the determination of sparfloxacin in human plasma and urine. The assay consisted of reversed-phase HPLC with ultraviolet detection. Plasma proteins were efficiently removed by precipitation with perchloric acid after the addition of grepafloxacin as an internal standard. For the urine samples, the only required sample preparation was dilution. Separation was achieved on a C18reversed-phase column. The quantification limit was 0.025 mg/L in plasma and 0.5 mg/L in urine. The coefficients of variation (CV) were less than 10% for intra-day and inter-day analyses. The recovery of sparfloxacin added to plasma and urine ranged from 96.7% to 97.9%. The method has been successfully applied to pharmacokinetic studies.