Widely used in enzymatic assays, particularly for measuring the activity of trypsin-like serine proteases. The compound acts as a chromogenic substrate, releasing yellow 4-nitroaniline upon cleavage by the target enzyme, which can be easily monitored spectrophotometrically at around 380–410 nm. Its high specificity makes it suitable for kinetic studies and inhibitor screening in biochemical and pharmaceutical research. Commonly employed in quality control of enzyme preparations and in high-throughput screening platforms due to its reliable signal output and stability under assay conditions.