Over the last decade sophisticated and powerful microcapillary HPLC for proteomic analysis have been developed increasingly and interfaced with high resolution tandem mass spectrometers. Separation prior to mass spectrometric (MS) analysis removes impurities, and concentrates analytes in the narrow elution peaks, resulting in increased sensitivity of MS analysis. This review will focus on the recent advances of on-line highperformance separation techniques based on microfluidic chips for complex proteomic analysis.