Reliable methods used to estimate the post-mortem interval (PMI) of skeletonized human remains are critically important for the accurate determination of time of death. During the early stages of decomposition (day ~ 1–25), PMI can be determined by visual observation, temperature analysis, and forensic entomology. On an annual timescale, luminol chemiluminescence and carbon isotope analysis can be used to determine the PMI. However, there is no reliable methodology that can be used for mid-term (day 22–300) timescale. In this study, we investigated the potential of Raman spectroscopy, a non-invasive and non-destructive analytical technique, for PMI analyses. The Raman-based diagnostics was achieved by spectroscopic analysis of human cadaver incisors. We observed gradual changes in the intensity of 1402 cm−1 band that can be used to differentiate days 22–42 and above day 42 PMI. These results demonstrate the great potential of Raman spectroscopy for precise and non-invasive analysis of decomposition processes in the human body which is critically valuable in forensic science.