Used as a chromogenic substrate in enzymatic assays, particularly for detecting esterase and lipase activity. Upon enzymatic hydrolysis, it releases 4-nitrophenol, which produces a yellow color detectable at 400–410 nm, enabling real-time monitoring of enzyme activity. Its dual nitrophenyl groups enhance electrophilicity, making it highly reactive toward nucleophilic attack by serine residues in hydrolases. Commonly employed in high-throughput screening, diagnostic kits, and biochemical research to quantify enzyme kinetics and inhibition. Also utilized in developing biosensors and studying enzyme mechanisms due to its clear colorimetric response.