Vitreous humor (VH) is a highly valuable sterile accessible sample in postmortem chemistry. Relatively few studies concerned the VH postmortem analysis in differentiating between causes of death. The current prospective cross-sectional study aimed at estimating VH postmortem levels of Sodium (Na), Potassium (K), Magnesium (Mg) and Glucose as differentiating variables between (traumatic, pathological and toxicological) causes of death. In the current work, VH samples were obtained from adult autopsied cases (n= 70) with postmortem interval less than 24 hours and after exclusion of ocular diseases or trauma. The traumatic group had significantly higher VH levels of (Na, K and Mg), in line with significantly lower VH glucose compared to Pathological group. The VH glucose levels were significantly higher in toxicological group compared to traumatic group. Toxicological group had significantly higher VH Na and VH Mg levels as well as significant lower VH glucose levels compared to pathological group. The VH glucose achieved the highest accuracy (84.6 %) in the receiver operating characteristic (ROC) curve analysis for differentiating between traumatic and pathological causes of deaths; followed by VHK, (82.6 %). Also, the VH glucose had the highest accuracy (76.2 %) in discriminating pathological from toxicological deaths followed by VH Na (70.8 %). Moreover, VH glucose achieved accuracy of (74.2 %) in differentiating between Traumatic and Toxicological deaths. To conclude, postmortem VH electrolytes and glucose levels showed variable patterns in different death causes. Hence, VH analysis could be used as adjuvant tool in suspecting cause of death; particularly the VH glucose level.