Simple SummarySnakebite envenoming is an important public health issue with annual mortality rates ranging between 81,000 and 138,000. Snake venoms may cause a range of pathophysiological effects and may have tissue-damaging activities that result in lifelong morbidities. The tissue-damaging components in snake venoms comprise multiple toxin classes with various molecular targets including cellular membranes and the extracellular matrix (ECM). In this study, we present multiple assay formats that enable us to study ECM degradation using a variety of fluorescently labeled ECM components. This workflow could provide valuable insights into the key mechanisms by which proteolytic venom components exert their effects. The workflow could prove useful for the development of effective snakebite treatments.AbstractSnakebite envenoming is an important public health issue with devastating consequences and annual mortality rates that range between 81,000 and 138,000. Snake venoms may cause a range of pathophysiological effects affecting the nervous system and the cardiovascular system. Moreover, snake venom may have tissue-damaging activities that result in lifelong morbidities such as amputations, muscle degeneration, and organ malfunctioning. The tissue-damaging components in snake venoms comprise multiple toxin classes with various molecular targets including cellular membranes and the extracellular matrix (ECM). In this study, we present multiple assay formats that enable investigation of snake venom-induced ECM degradation using a variety of (dye-quenched) fluorescently labeled ECM components. Using a combinatorial approach, we were able to characterise different proteolytic profiles for different medically relevant snake venoms, followed by identification of the responsible components within the snake venoms. This workflow could provide valuable insights into the key mechanisms by which proteolytic venom components exert their effects and could therefore prove useful for the development of effective snakebite treatments against this severe pathology.