A sensitive high performance liquid chromatographic method for the determination in biological fluids of 1-cyclopropyl-6-fluoro-1,4- dihydro-8-methoxy-7-(3-methylaminopiperidin-1-yl)-4-oxoquinoline-3 -carboxylic acid dihydrate (CAS 127294-70-6, balofloxacin, Q-35), a new fluoroquinolone, as well as its metabolites, has been developed. Balofloxacin and its metabolite, N-desmethyl balofloxacin, were separated and determined by reversed-phase column with fluorescence detection. Balofloxacin glucuronide was determined as balofloxacin after alkali hydrolysis. This method exhibited good precision and accuracy. Furthermore, it is a simple method and requires only a small amount of samples, and therefore should be useful for pharmacokinetic studies in animals and humans.