A high-performance liquid chromatographic method has been developed in normal-phase conditions for the separation of enantiomeric gatifloxacin, (+/-) 1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-quinoline-3-carboxylic acid, an antibiotic in bulk drug. The method involved the use of an amylose-based Chiralpak AD-H (150 mm x 4.6 mm, 5 microm) column using a mobile phase system containing n-hexane-ethanol-diethylamine (85:15:0.1% v/v). The conditions affording the best resolution were found by selection and variation of the mobile-phase compositions and the differences in separation capability of the method is noted. Relative standard deviation of retention times and peak areas were better than 0.2% and 0.4%, respectively, for precision. Gatifloxacin sample solution and mobile phase are found to be stable for at least 48 h.