Updated project metadata. Ancient protein analysis provides clues to human life and diseases from ancient times. Paleoproteomics has the potential to give a better understanding of the modes of fabrication of ancient materials, their composition, and pathways of degradation, as well as the development of animal fibers through domestication and breeding. Thus, this study aimed at providing guidance for choosing proteomics workflows to analyze leather samples and their capacity to distinguish between unknown archeological species. Here, we performed shotgun proteomics of archeological animal skin for the first time. The raw output data were analyzed using three different software (Proteome Discoverer, Protein Pilot, and Peptide Shaker) with their impeded algorithms. The study found that the best species identification percentage was obtained using protein piolet with protein database. Particularly prevalent and relatively high collagen expression suggests its resistance to degradation, despite the samples’ exposure to environmental and chemical alterations. The success of this case study indicates that further analyses could assist in reworking historical baseline data for putative identification of unknown archeological samples.