Used as a chromogenic substrate in enzymatic assays, particularly for the detection and measurement of acylase enzyme activity. Upon enzymatic hydrolysis, it releases 4-nitroaniline, which produces a yellow-colored solution in alkaline conditions, allowing for spectrophotometric detection at around 380–405 nm. This makes it valuable in biochemical research and diagnostic applications where sensitive and quantitative enzyme activity monitoring is required. Its specificity for certain acylases enables selective analysis in complex biological samples.