L-Leucine-p-nitroanilide is primarily used as a substrate in enzymatic assays to study the activity of proteases, particularly leucine aminopeptidases. These enzymes play a crucial role in breaking down proteins and peptides, and the compound helps in detecting and quantifying their activity. When the enzyme cleaves the amide bond in L-Leucine-p-nitroanilide, it releases p-nitroaniline, which is a yellow-colored product. This color change can be easily measured using spectrophotometry, providing a straightforward method to monitor enzyme kinetics and efficiency. Its application is significant in biochemical research, drug development, and clinical diagnostics, where understanding protease activity is essential.