A method used for determining the enantiomeric excess (ee) value of chiral amino compounds by MEKC is described. In this method, the plug-plug type electrophoretic medicated microanalysis technique was employed to convert the enantiomers of chiral amino compounds into their diastereomers through an on-column derivatization with o-phthaldialdehyde and the chiral reagent N-acetyl-L-cysteine. Afterwards, the resulting diastereomers were easily separated with a nonchiral MEKC approach. The on-column derivatization conditions and the separation conditions were optimized and the method was validated with five chiral amino compounds. The present method can be used for assaying the ee value of chiral amino compound with various structural features, especially for those that have no UV chromophore. Therefore, the method can be potentially used for screening or evaluation of the asymmetric catalysts developed by the combinatorial chemistry. In this case, the ee values of chiral products with various structures need to be measured; however, this is difficult for direct chiral separation approach due to the fact that the chiral selectivity is strongly dependent on the structure of the analytes. The method is simple, reliable, and automatic.