Cellular processes such as tissue regeneration, inflammation, and migration require the proteolysis of the extracellular matrix and the proteolytic activation of signaling molecules. A widely used and accessible technique for studying these processes is gelatin zymography, particularly for investigating matrix metalloproteinases (MMPs), though it is not limited to them. This method is favored for its simplicity, low cost, and robustness. Despite certain limitations, it remains a preferred approach for the initial investigation of complex samples. Here, we present a protocol applicable to various sample sources, including proteases from human cell lines and bacteria isolated from chronic wounds. We also explore changes in protease activity within exudates from human chronic wounds, a challenging analysis for more complex techniques. Additionally, we emphasize the potential to extend the basic protocol to study the conditions under which proteases are active.