Abstract: A continuous flow method has been developed for the automatic determination of enterokinase in rat small intestine mucosa and/or luminal content. Trypsinogen was first hydrolyzed by enterokinase under conditions which minimize autocatalytic activation. L-benzoyl-arginine paranitroanilide was then added and split to paranitroaniline by the trypsin so formed. Liberated paranitroalinine was diazotized and converted by the Bratton-Marshall reagent (N-naphthyl ethylene diamine) to an azodye, with maximum absorption at 550 nm. This method of determination was found to be six times more sensitive than the direct p-nitroaniline determination method. 36 determinations can be made hourly.