Post-mortem interval (PMI) estimation remains a challenge for forensic pathologists, thus representing a hot topic in the research of forensic sciences. Over recent years, new methods have been proposed based on the analysis of various human biological matrices and changes in their post-mortem biochemical markers (BPMs) concentrations. Raman spectroscopy shows great promise in PMI assessment thanks to its ability to analyze complex organic materials and provide a unique fingerprint of substances. Following PRISMA guidelines, this systematic review addresses the PICO question: “In human cadavers/remains, what is the role of Raman spectroscopy, on what biological matrices, and with what analytical techniques is it currently applied for post-mortem interval estimation?“. The selected five papers reviewed point out that the applications of Raman spectroscopy in PMI estimation are now widespread in forensic anthropology, moving from the study of evolving chemical alterations of human skeletal remains over time to exploring their correlation with burial duration or PMI. The most frequently evaluated parameters were phosphate (an inorganic component of the bone/tooth matrix) and the progressive decrease in the organic bands’ peak intensity over PMI, with principal component analysis (PCA) being the preferred multivariate statistical analysis to improve the interpretation of Raman spectral data. Although Raman spectroscopy has many analytical and practical advantages, being non-destructive, rapid, and available as an hand-held device, its implementation in forensic routine casework remains limited and can only be expected to be useful in cases with longer PMI.