This compound is primarily used in biochemical research as a substrate for various proteolytic enzymes, particularly those involved in blood coagulation and fibrinolysis. It is often employed in assays to measure the activity of enzymes like thrombin, plasmin, and trypsin. The cleavage of the peptide bond by these enzymes releases 4-nitroaniline, which can be detected spectrophotometrically at 405 nm, allowing for quantitative analysis of enzyme activity. Its specificity and sensitivity make it a valuable tool in studying enzyme kinetics, inhibitor screening, and understanding mechanisms of proteolysis in biological systems. Additionally, it is utilized in clinical diagnostics to assess coagulation disorders and monitor therapeutic interventions.