Abstract: This paper introduced a method that based on the reaction of primary amino groups with o-phthaldialdehyde (OPA) for determining the hydrolytic degree of cottonseed protein.Neutral protease was used to hydrolyze cottonseed protein and the degree of hydrolysis (DH) was compared with three assay methods, i.e.o-phthaldialdehyde method, formaldehyde titration method and ninhydrin colorimetric method.Comparing with formaldehyde titration and ninhydrin colorimetric methods, results showed that OPA method performed more accurate and faster in analyzing, easier to handle, repeatable in results and friendly for environment.It is recommended that OPA method is an appropriate method for evaluating in hydrolytic degree of cottonseed protein.