The presence and activity of matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) in dentin significantly affect the progress of carious and erosive lesions into dentin, as well as the durability and integrity of dental restorations. Understanding the mechanisms involved on this proteolytic activity could therefore provide valuable insights into the pathogenesis of these conditions and inform the development of novel therapeutic strategies. This manuscript provides a comprehensive overview of zymographic techniques applied in dental research, with a focus on the sample’s preparation and different measurements of the enzymatic activity through substrate hydrolysis. Standard zymography assays using dentin as a substrate to evaluate the gelatinolytic activity of metalloproteinases (MMPs) and cysteine cathepsins (CCs) are presented, including gel zymography, in situ zymography using fluorescent gelatin-substrate, and reverse zymography for investigating proteolytic activity in dentin.