L-Proline 4-nitroanilide is primarily used as a substrate in enzymatic assays to study the activity of proline-specific enzymes, such as prolyl endopeptidases. These enzymes play a significant role in various biological processes, including peptide metabolism and neuropeptide regulation. In research, it helps in understanding enzyme kinetics and inhibition mechanisms, which can be crucial for developing therapeutic agents targeting enzyme-related disorders. Additionally, its application extends to biochemical studies where it aids in identifying and characterizing enzyme specificity and function.