Zymography is a powerful technique for detecting and identifying proteolytic enzyme activity through gel electrophoresis based on molecular weights determined by substrate degradation. Gelatin zymography specifically detects proteolytic enzymes that degrade gelatin, such as matrix metalloproteinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B). In contrast, plasminogen-casein zymography detects plasminogen-dependent serine proteases, such as tissue-type plasminogen activator and urokinase plasminogen activator. Various cells, tissues, and fluids can be assayed using these techniques, with methodologies for collecting and processing reproductive fluids to accurately detect protease activities detailed herein. General considerations for performing these experiments, including major troubleshooting and safety issues, are also outlined. Proper interpretation of gelatin and plasminogen-casein zymography data requires a thorough understanding of the technique’s principles and pitfalls, which is crucial when evaluating enzyme levels and the presence of active gelatinase species and plasminogen-dependent proteases. When properly utilized, gelatin and plasminogen-casein zymography are excellent tools for studying metalloproteases and serine proteases in different biological systems.