<p>Postmortem interval (PMI) estimation is a critical component of forensic investigations as it can help establish a timeline of events and assist in reducing a pool of missing persons or potential suspects. Despite its importance, the accuracy of PMI estimation remains challenging due to the complexity of decomposition and limitations of current methods, particularly in the later postmortem period. Proteomics has shown potential as a more objective method, with current studies taking an untargeted approach to identify informative peptide targets. Previous studies in our laboratory analysed human muscle tissue and identified 12 peptide ratios, consisting of 19 individual peptides, that were correlated with decomposition time. This study aimed to optimise the detection of these 19 peptide targets using a targeted liquid chromatography triple quadrupole mass spectrometry (LC-QQQ) method suited for casework applications and instrument platforms commonly found in forensic laboratories. Of the 19 peptide targets, 17 were optimised to be reproducible. Additionally, a longitudinal analysis in authentic human samples was conducted over a 14-day period postmortem to monitor detection throughout the decomposition process. All but one of the targets produced interpretable results, and data analysis was performed to generate peptide ratios. After log2 transformation, two of the previously proposed peptide ratios showed linear trends. Overall, this study has shown that a targeted LC-QQQ method is a viable system for the detection of PMI peptide targets from human postmortem muscle tissue and has demonstrated the potential for this type of analysis to be validated and applied in routine forensic casework.</p>