Monolithically integrated, polymer (SU-8) microchips comprising an electrophoretic separation unit, a sheath flow interface and an ESI emitter were developed to improve the speed and throughput of proteomics analyses. Validation of the microchip method was performed based on peptide mass fingerprinting and single peptide sequencing of selected protein standards. Rapid, yet reliable identification of four biologically important proteins (cytochrome C, beta-lactoglobulin, ovalbumin and BSA) confirmed the applicability of the SU-8 microchips to ambitious proteomic applications and allowed their use in the analysis of human muscle cell lysates. The characteristic tryptic peptides were easily separated with plate numbers approaching 10(6), and with peak widths at half height as low as 0.6 s. The on-chip sheath flow interface was also exploited to the introduction of an internal mass calibrant along with the sheath liquid which enabled accurate mass measurements by high-resolution Q-TOF MS. Additionally, peptide structural characterization and protein identification based on MS/MS fragmentation data of a single tryptic peptide was obtained using an ion trap instrument. Protein sequence coverages exceeding 50% were routinely obtained without any pretreatment of the proteolytic samples and a typical total analysis time from sampling to detection was well below ten minutes. In conclusion, monolithically integrated, dead-volume-free, SU-8 microchips proved to be a promising platform for fast and reliable analysis of complex proteomic samples. Good analytical performance of the microchips was shown by performing both peptide mass fingerprinting of complex cell lysates and protein identification based on single peptide sequencing.