Abstract Introduction The estimation of post-mortem interval (PMI) remains a major challenge in forensic science. Most of the proposed approaches lack the reliability required to meet the rigorous forensic standards. Objectives We applied ¹H NMR metabolomics to estimate PMI on ovine vitreous humour comparing the results with the actual scientific gold standard, namely vitreous potassium concentrations. Methods Vitreous humour samples were collected in a time frame ranging from 6 to 86 h after death. Experiments were performed by using ¹H NMR metabolomics and ion capillary analysis. Data were submitted to multivariate statistical data analysis. Results A multivariate calibration model was built to estimate PMI based on 47 vitreous humour samples. The model was validated with an independent test set of 24 samples, obtaining a prediction error on the entire range of 6.9 h for PMI < 24 h, 7.4 h for PMI between 24 and 48 h, and 10.3 h for PMI > 48 h. Time-related modifications of the ¹H NMR vitreous metabolomic profile could predict PMI better than potassium up to 48 h after death, whilst a combination of the two is better than the single approach for higher PMI estimation. Conclusion The present study, although in a proof-of-concept animal model, shows that vitreous metabolomics can be a powerful tool to predict PMI providing a more accurate estimation compared to the widely studied approach based on vitreous potassium concentrations.