Abstract: The activity, or concentration, of a hydrolyzing enzyme, e.g., an endo-enzyme, is determined by providing a detection layer comprising a carrier matrix and a water-insoluble substrate dispersed in said carrier matrix which substrate is detectably marked, e.g., dyed, and which substrate, upon action of a hydrolyzing enzyme thereon, is broken down into soluble fragments capable of diffusing through the carrier matrix to cause formation of an unmarked area in said detection layer; and measuring the size of the unmarked area as a measure of the activity of concentration of said enzyme.