In the framework of the C-HPP, our Franco-Swiss consortium has adopted chromosomes 2 and 14, coding for a total of 382 missing proteins (proteins for which evidence is lacking at protein level). Over the last four years, the French proteomics infrastructure has collected high quality datasets from 40 human samples, including a series of rarely studied cell lines, tissue types and sample preparations. Here, we described a step-by-step strategy based on the use of bioinformatics screening and subsequent mass spectrometry (MS)-based validation to identify what were up to now missing proteins in these datasets. Screening database search results (85,326 .dat files) identified 58 of the missing proteins (36 on chromosome 2 and 22 on chromosome 14) by 83 unique peptides following the latest release of neXtProt (2014-10). PSMs corresponding to these peptides were thoroughly examined by applying two different MS-based criteria: peptide-level False Discovery Rate (FDR) calculation and expert PSM quality assessment. Synthetic peptides were then produced and used to generate reference MS/MS spectra. A spectral similarity score was then calculated for each pair of reference-endogenous spectra and used as a third criterion for missing protein validation. Finally, LC-SRM assays were developed to target proteotypic peptides from 4 of the missing proteins detected in tissue/cell samples which were still available and for which sample preparation could be reproduced. These LC-SRM assays unambiguously detected the endogenous unique peptide for 3 of the proteins. For 2 of these, identification was confirmed by additional proteotypic peptides. We concluded that of the initial set of 58 proteins detected by the bioinformatics screen, the consecutive MS-based validation criteria led to propose the identification of 13 of these proteins (8 on chromosome 2 and 5 on chromosome 14) that passed at least two of the three MS-based criteria. Thus, a rigorous step-by-step approach combining bioinformatics screening and MS-based validation assays is particularly suitable to obtain protein-level evidence for proteins previously considered as missing. All MS/MS data have been deposited in ProteomeXchange under identifier PXD002131.