Here we report our approach to the peptidomic analysis of mouse liver. We use ultrafiltration for peptide prefractionation, which is followed by size exclusion chromatography. The low molecular weight peptides (MW below ~3 kDa) are analysed directly by nanoLC-MS/MS, and the higher molecular weight peptides (MW above ~3 kDa) are characterized with MALDI-TOF MS first and then proteolytically digested prior to nanoLC-MS/MS analyses.