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The estimation of the time since death, or post-mortem interval (PMI), still remains a main conundrum in forensic science. Several approaches have been so far proposed from either a qualitative or a quantitative point of view, but they still lack reliability and robustness. Recently, metabolomics has shown to be a potential tool to investigate the time-related post-mortem metabolite modifications in animal models.Here we propose, for the first time, the use of aAH samples were collected at different times after death (from 118 to 1429 min).A multivariate calibration model was built to estimate PMI on the basis of the metabolite content of the samples. The model was validated with an independent test set, obtaining a prediction error of 59 min for PMI 500 min, 104 min for PMI from 500 to 1000 min, and 118 min for PMI 1000 min. Moreover, the metabolomic approach suggested a picture of the mechanisms underlying the post-mortem biological modifications, highlighting the role played by taurine, choline, and succinate.The time-related modifications of the