Chitinases are a variety of different enzymes that facilitate the breakdown of chitin, a complex sugar molecule presents in the outer shells of arthropods and the cellular walls of fungi. Chitinase zymography is a powerful analytical technique to visualize and characterize chitinase activity in complex biological samples. In plants, chitinases contribute to defense mechanisms by degrading fungal cell walls and deterring pathogen invasion. They also influence plant growth and development by modulating interactions with soil microbes. This chapter provides different zymographic protocols for chitinase detection, including those using polyacrylamide gel electrophoresis (PAGE) with various chitin derivatives and staining methods. Calcofluor White M2R, is offering unique advantages for visualizing chitinase activity on native PAGE overlayed chitin amended gels. This overview highlights the importance of selecting appropriate substrates and assay conditions to achieve accurate and reliable results in chitinase zymography, thereby advancing the potential of chitinase-based technologies in industrial and environmental fields.