In this work, a novel multiway approach by spectroscopy and thermogravimetry associated with chemometrics is developed, providing a multiparametric characterization of vitreous humor as a function of the time since death. Estimation of the precise time since death was performed on hospital deaths occurred in casualty, by medico-legal and post mortem examination, with no metabolic disorders. Micro and macro elements in vitreous specimens were determined by ICP-OES and were found to be diagnostic in predicting the post mortem interval (PMI). The percentage of bulk and bound water provided by thermal analysis investigation was correlated to spectroscopic analysis, and chemometric tools were used to compare results and to develop a model of prediction of PMI. The study reveals a significant role of P, S, and Mg in addition to the potassium concentration in determining the death interval and permits increased accuracy with respect to conventional procedures and allows the investigation of PMI to be extended to 15 days.