L-Alanine 4-nitroanilide hydrochloride is primarily used in biochemical research as a substrate for studying enzyme activity, particularly proteases and peptidases. It serves as a chromogenic substrate, enabling the detection and quantification of enzyme activity through colorimetric assays. When the enzyme cleaves the compound, it releases 4-nitroaniline, which produces a yellow color that can be measured spectrophotometrically. This makes it a valuable tool in enzymology for investigating enzyme kinetics, inhibition, and specificity. Additionally, it is utilized in diagnostic applications to assess enzyme function in clinical and laboratory settings.