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In this study, a capillary electrochromatography (CEC) coupled with mass spectrometry (MS) detection was used for penicillins separation.Monolithic stationary phases prepared by a single step in situ polymerization were developed as separation columns.In this step we got different effect with different charged monomer.In order to achieve good separation efficiency and sensitivity on CEC and CEC-MS, several parameters such as the composition of monomer mixtures, composition and the volume fraction ratio of ACN in sample matrix were optimized.Furthermore, online sample concentration steps were used to enhance the detection sensitivity.This work demonstrated that the CEC method coupled with MS detector could achieve reproducible baseline separation for penicillins with good detection limit.