L-Phenylalanine 4-Nitroanilide is primarily used as a substrate in enzymatic assays to study the activity of proteolytic enzymes, particularly those that cleave peptide bonds. It is often employed in biochemical research to investigate the specificity and kinetics of enzymes like chymotrypsin and other serine proteases. The compound releases a colored product upon enzymatic cleavage, making it useful for spectrophotometric detection and quantification of enzyme activity. This application is critical in understanding enzyme mechanisms, screening enzyme inhibitors, and developing diagnostic tools for enzyme-related disorders. Additionally, it serves as a valuable tool in pharmaceutical research for drug development targeting specific proteases.