Abstract A highly sensitive and accurate automated method using a Technicon Auto-analyzer AAII was developed for the determination of human serum γ-glutamyl transpeptidase. γ-L-Glutamyl- p-nitroanilide added as substrate was split to p-nitroaniline and the γ-glutamyl group was trapped by glycylglycine in Ammediol/HCl buffer solution. Liberated p-nitroaniline was diazotized and converted by Tsuda Reagent ( N, N-(diethyl)- N′-(l-naphthy1)ethylendiamine) to an azo-dye possessing an absorbance maximum at 550 nm. This new method of determination, using the conversion of p-nitroaniline to azo-dye, was found to be 7 times more sensitive as compared to the conventional direct p-nitroaniline determination method . This method requires only 0.1 ml serum and permits 60 determinations per hour.