A new micro-method for the assay of hyaluronidase activity is described. The method utilizes chondroitin sulfate as a substrate. The degraded products of chondroitin sulfate by hyaluronidase are determined by modified disc gel electrophoresis. The products are first concentrated into a single band in acrylamide gel, and then the band is stained with cetylpyridinium chloride. The absorbance of the band is proportional to the amount of the degraded products. The hyaluronidase activity is therefore linearly related to the absorbance. This procedure represents a sensitive method for the assay of hyaluronidase activity in the serum and urine.