Proteases or peptidases are hydrolases that break down polypeptide chains into smaller peptide subunits or amino acids. The search for proteases is important and often required using the sensitive screening technique known as zymography. Zymography is a two-stage technique that involves separating proteins, via electrophoresis, followed by the detection of protease activity. After the release of free peptides or amino acids from a protein substrate, a clear zone is formed in this protease screening method. A typical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer is used to prepare the samples, without reducing conditions. Boiling or reducing agents are not required because they would prevent the enzyme from refolding. Here, we discuss the need for standardized sensitivity and detection metrics in the various zymographic methods, and we emphasize the value of protease zymography for the detection and in-depth characterization of microbial proteases in enzymology research.