Abstract: Novel phenylazonaphthol derivatives, e.g., 4-(4'-nitro-2'-sulfophenylazo)-1-naphthyl-N-acetyl-.beta.-D-glucosaminide sodium salt, are excellent substrates for determining N-acetyl-.beta.-D-glucosaminidase (NAG) activity in urine collected from the patient with renal disorders. The substrate has high water solubility, is not affected by interferences in urine and provides a simple method for determination of NAG activity which is suited for both rate and endpoint assays.