Updated project metadata. Colorectal cancer (CRC) is one of the most prevalent and lethal cancer diseases worldwide. Here, we aimed to identify and quantify CRC serum biomarkers by combining a robust label-free quantification procedure followed of two consecutive steps of targeted parallel reaction monitoring (PRM) for biomarker validation in a fully inclusive proteomic strategy. For the discovery phase pooled serum samples were used for shotgun proteomics and label-free quantification. On the identification phase, 116 potential biomarkers were selected based on their statistical significance and their relative expression in disease stages respect to healthy stage and their functional relation with cancer progression. Verification phase was conducted in 2 steps. In the first step, 318 peptides from 116 proteins were used for PRM verification giving place to 23 PRM-quantifiable, potential CRC biomarkers. In a second step, 7 peptides corresponding to CO9, APOC3, CRP, THSB1, ECM1 and IGF2 proteins were reproducibly confirmed by PRM in every CRC stage for these unfractionated samples. Finally, a different cohort composed by individual serum samples was used in the final validation phase. In individual serum samples, 5 peptides belonging to 4 proteins were consistently quantified and validated. ROC analyses indicated that peptides GWVTDGFSSLK and LCNNPTPQFGGK were suitable candidates for predicting the separation between control and CRC patients. Two assays for absolute quantification of significant peptides in serum samples were established using AQUA peptides. In conclusion, a set of serum peptides were validated by PRM as potential biomarkers for differentiating control from CRC patients.