Estimation of the postmortem interval (PMI) has attracted the attention of many researchers. It is generally accepted as a challenging task in forensic medicine. Due to its difficulty, researchers have tried to estimate the PMI using different physical and chemical techniques. Since the PMI estimation accuracies of previous studies are not at the desired level, new methods should be developed to more accurately estimate the PMI. The development of opacity in the eye in the PMI might be an important breakthrough in this field. After death, corneal hydration occurs due to degenerated endothelial cells. The degenerated endothelial barrier of the cornea cannot prevent the flow of aqueous humor to the cornea, which results in opacity. The amount of aqueous humor in the cornea determines the level of opacity. Since the flow of aqueous humor to the cornea will continue for a while, opacity is expected to increase with the PMI. In this study, images of human eyes were investigated using computer-based image analysis. The corneal and non-corneal opacities of the recorded eye images increase during the experiment. The experimental results prove that there is a correlation between the elapsed time after death and the development of opacity in the corneal and non-corneal regions in human cases. Exponential curve fitting is employed to observe the decay of the opacity over time. A repeated ANOVA test is also used to show that the opacity development is statistically significant. (C) 2017 Nalecz Institute of Biocybernetics and Biomedical Engineering of the Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.