A rapid, sensitive and simple procedure has been developed for determination of argininosuccinate (ASA). The assay is based upon the enzymatic cleavage of ASA in the presence of excess of argininosuccinate lyase (ASAL), HPLC separation and UV-detection of fumarate formed. The calibration curve, obtained using standards, derives from a linear correlation between the reaction rate and ASA concentration. The range of HPLC detection corresponds with the level of normal excretion of argininosuccinate in urine from the patients with argininosuccinate lyase deficiency (ASLD).