Objective — evaluation of the relationship between the activity of proteolytic enzymes (cathepsin D and calpains) and the dynamics of the fluorescence intensity of the coenzymes nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in the rats’ skeletal muscles in relation to the time of death. The proteolytic activity of enzymes in rat skeletal muscle was determined at the postmortem time points corresponding to the most significant changes in the dynamics of coenzymes NADH and FAD fluorescence intensity. The proteolytic enzymes activity was found to be low during the period of increasing intensity of NADH fluorescence observed within 3 hours after death. An increase in the activity of proteolytic enzymes was registered in 4.5 hours after death which corresponds to the initial point of decrease in NADH fluorescence intensity. In 24 hours post-mortem, corresponding to increased FAD fluorescence intensity a significant decrease in the activity of calpains was found. The results of the study suggest that the nature of the postmortem dynamics of the fluorescence intensity of coenzymes is largely due to the peculiarities of intracellular proteolysis. The study results suggest that the pattern of post mortem changes in coenzyme fluorescence intensity is largely attributable to the specifics of intracellular proteolysis. The relationship between coenzyme fluorescence and molecular mechanisms of cell death confirms the viability and feasibility of laser-induced spectroscopy for post-mortem changes assessment when determining the time of death. © 2021, Media Sphera Publishing Group. All rights reserved.