A simple, accurate and precise high-performance liquid chromatographic method was developed and validated for the determination of trovafloxacin, a new quinolone antibiotic, in serum and urine. Following solid-phase extraction, chromatographic separation was accomplished using a C18 column with a mobile phase consisting of 0.04 M H3PO4-acetonitrile-tetrabutylammonium hydroxide-0.005 M dibutyl amine phosphate (D-4) reagent (83:16.85:0.05:0.1, v/v), pH 3. Trovafloxacin and the internal standard (a methyl derivative of trovafloxacin) were detected by ultraviolet absorbance at 275 nm. The lower limit of quantification for trovafloxacin was 0.1 microgram/ml and the calibration curves were linear over a concentration range of 0.1 to 20.0 micrograms/ml (r2 = 0.9997). The average recoveries were greater than 70% for both trovafloxacin and internal standard. The intra-day and inter-day coefficients of variation were generally less than 5% in urine and serum over the concentration range of 0.1 to 20.0 micrograms/ml. Human serum samples could be stored for up to 12 months at -20 degrees C and urine samples could be stored up to 18 months at -80 degrees C.