We applied a metabolomic approach to monitor the modifications occurring in goat vitreous humor (VH) metabolite composition at different times (0, 6, 12, 18, and 24 hours) after death. The (1)H-NMR analysis of the VH samples was performed for the simultaneous determination of several metabolites (i.e., the metabolite profile) representative of the VH status at different times. Spectral data were analyzed by Principal Component Analysis (PCA) and by Orthogonal Projection to Latent Structures (OPLS) regression technique. PCA and OPLS suggested that different spectral regions were involved in time-related changes. The major time-related compositional changes, here detected, were the increase of lactate, hypoxanthine, alanine, total glutathione, choline/phosphocholine, creatine, and myo-inositol and the decrease of glucose and 3-hydroxybutyrate. We attempted a speculative interpretation of the biological mechanisms underlying these changes. These results show that multivariate statistical approach, based on (1)H NMR metabolite profiling, is a powerful tool for detecting ongoing differences in VH composition and may be applied to investigate several physiological and pathological conditions.