Multidimensional protein identification technology (MudPIT) is one of the most versatile methods for separating and identifying highly complex peptides or proteins. However, there are still inherent problems resulting from salt in eluent systems and instrumentation with MudPIT. We designed a novel and simple flow path using twovalve system and successfully performed a fully automated peptide analysis using MudPIT coupled with nano-liquid chromatography electrospray ionization mass spectrometry (nLC-ESI-MS). It enables to generate a remarkably stable nanospray during the MudPIT analysis and realize the fully automated MudPIT system. This column arrangement could be easily installed to avoid laborious loading steps and unstable ionization from discontinuous flow. Consequently, the new flow path design for MudPIT system guarantees the detection of more peptides and higher protein coverage in proteome analysis.