H-D-Phe-Pip-Arg-pNA hydrochloride, a chromogenic substrate, mimics the N-terminal segment of the A alpha chain of fibrinogen, the native substrate of thrombin. It displays specificity towards thrombin and is employed for quantifying antithrombin-heparin cofactor (AT-III). The utilization of H-D-Phe-Pip-Arg-pNA hydrochloride in the AT-III assay enables a sensitive, accurate, and straightforward measurement process.