Analysis of biochemical constituents of the vitreous humor can be useful in determining the postmortem interval as there is proportionate postmortem rise of potassium and fall in sodium concentration. We studied 120 autopsy cases to determine the utility of potassium, sodium, calcium, and chloride levels, and sodium/potassium ratio in estimating the postmortem interval. There was a linear relationship between vitreous potassium concentration and postmortem interval, whereas an inverse relationship between vitreous sodium/potassium ratio and postmortem interval was noted. Other factors like age, sex, cause of death, season of death, and refrigeration of sample did not influence the vitreous humor potassium values. Using the statistical tools, a new formula was derived to determine the postmortem interval based on the potassium concentration and a review of previous literature is presented. Hence, the findings of this study supported a central role of vitreous humor biochemistry in many postmortem forensic and pathological evaluations.