Colorectal cancer (CRC) is the second most common cause of cancer-related deaths worldwide with an annual incidence of almost a million cases and an annual mortality around 500 000. The fecal occult blood test is currently the first line method for CRC screening, but has unacceptably low sensitivity and specificity. Improved screening tests are therefore urgently required for early-stage CRC screening when therapy is most likely to be effective. We describe a discovery-based proteomics hypothesis using orthogonal multi-dimensional fractionation (1-D SDS-PAGE, RP-HPLC, size exclusion chromatography) to mine deep into the fecal proteome for the initial discovery process, which generated a library containing 108 human fecal proteins with the associated peptide and MS/MS data. These data were then used to develop and optimize a multiplex multiple reaction monitoring assay for 40 non-redundant human proteins present in the feces. To show proof of principal for clinical analysis, multiplex screening of these 40 proteins was carried out on fecal samples from eight CRC patient and seven normal volunteers. We identified 24 proteins consistently found in all samples and nine proteins found only in the CRC patients, showing the potential of this approach for the analysis of potential CRC biomarkers. Absolute quantitation using C-terminal isotopically labeled synthetic peptides corresponding to hemoglobin and carcinoembryonic antigen 5 was also performed.