A method used for quantitative analysis of bacitracin with micellar electrokinetic capillary chromatography (MEKC) is described. As capillary zone electrophoresis gave poor separation selectivity, MEKC was preferable. It was found that a zwitterionic surfactant, 3-(N,N-dimethylhexadecylammonium)-propanesulfonate (PAPS) gave the best selectivity among the several surfactants studied. As the analytes tend to adsorb onto the capillary wall due to their positive charge, an acidic solution composed of Tris-phosphate buffer at pH 2.5 was necessary to diminish such adsorption. The peak tailing caused by relatively strong ion pair interaction between the analyte and PAPS micelle could be reduced by adding nonionic surfactant Brij 35 to the PAPS solution. This phenomenon is possibly explained by a mixed micelle mechanism. In order to obtain the optimal conditions and to test the method robustness, a central composite experimental design was performed. The optimal conditions are as follows: 44 cm length of fused-silica capillary with 50 microm inner diameter, 90 mM Tris-phosphate buffer (pH 2.5) containing 17 mM PAPS and 0.3% w/v-Brij 35, 18 kV applied voltage, UV detection at 192 nm and 25 degrees C column temperature. Under the optimal conditions, more than 50 peaks could be obtained in 30 min. The method had a linearity range from 1 to 0.05 mg/mL (concentration of bacitracin A). The limit of quantitation (LOQ) and limit of detection (LOD) were 0.005 and 0.0012 mg/mL, respectively.