Hyaluronan and hyaluronidases are critical in tissue remodeling, inflammation, and tumor progression. This chapter provides a comprehensive guide to hyaluronan zymography, a powerful technique for detecting and quantifying hyaluronidase activity in complex biological samples. The method involves separating proteins by polyacrylamide gel electrophoresis with hyaluronan incorporated into the gel matrix. Following electrophoresis, the gel is incubated to allow hyaluronidases to degrade the hyaluronan substrate, resulting in clear digestion zones. Detailed protocols for sample preparation and the zymographic process are included, offering researchers a robust tool for studying hyaluronidase activity and regulation in various biological contexts.