Background: Estimation of the postmortem interval (PMI) is a pivotal task in forensic investigations. Methods relied on PM biochemical alteration of the compounds such as cadaverine (Cad) and putrescine (Put) can provide more accurate data about the exact PMI. Aim: To investigate the correlation between Cad and Put levels in human brain tissue, measured by gas chromatography-mass spectrometry (GC-MS), and the PMI, to see if they can be used as reliable indicators for PMI estimation. Methodology: Brain specimens were taken during autopsy of three male victims, who died by stab wounds. Autopsy was done within three hours after death (the moment of death was known). The fifteen brain specimens (five from each victim’s brain, according to a fixed time schedule to simulate progressive postmortem intervals), were prepared for injection into the GC-MS and the levels of Cad and Put in the brain tissue specimens were measured. Results: Both Cad and Put levels increased in the brain tissue samples as long as the PMI increased, and there was a statistically highly significant (p |