Updated publication reference for PubMed record(s): 33453434. The proteomics of archival samples has advanced significantly during the last two decades, making possible the use of vast FFPE tissue archives in biomarker studies. However, due to the limited data regarding comparison of the performance of different protocols, there is currently no consensus on the most effective protocol for shotgun proteomic analysis of FFPE tissue. In-solution digestion method using Rapigest as detergent and FASP method, both reported to deliver superior results on FFPE tissues were compared using two label-free data-independent LC-MS/MS acquisition modes. Archival FFPE prostate tissues stored 7 years and mirroring fresh frozen samples were processed with both protocols in 3 technical replicates. Data were comparatively evaluated in terms of protein and peptide identifications in fresh frozen and FFPE samples, analysis of the fresh and FFPE tissues representative proteome (molecular weight distribution, distribution according to protein’s pI, cellular component analysis of proteins and quantitation accuracy) and technical reproducibility of the Rapigest and FASP protocols.