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The discovery of new methods for determining the post-mortem interval is of significant forensic interest. Mass spectrometry has enhanced the accuracy of assessing post-mortem protein decay, with skeletal muscle being the most studied substrate due to its intrinsic properties of postmortem decay. In this pilot study, human skeletal muscle tissue (iliopsoas) was harvested and allowed to decay under controlled temperature and humidity conditions at predetermined intervals. The samples were analyzed using mass spectrometry proteomics for both qualitative and quantitative evaluation of proteins and peptides. Candidate proteins were validated through immunoblotting. The results were significant, identifying several proteins that could aid in estimating the post-mortem interval. Notably, PLIN4, MYOZ2, SYNPO2, and BAG3 were validated by immunoblotting over a broader range of experimental points and temperatures. Furthermore, human results were compared with animal muscle samples from a previous study, revealing similarities in decomposition kinetics. This analysis of human samples marks a step forward in the potential forensic application of proteomic evaluation by mass spectrometry., Competing Interests: Declaration of Competing Interest The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)