Used as a substrate in enzyme activity studies, particularly for esterases and lipases, where its hydrolysis produces 4-nitrophenol, a yellow-colored product that can be easily monitored spectrophotometrically at 400-410 nm. Commonly employed in kinetic assays to measure enzyme efficiency and inhibition. Also utilized in biochemical research for detecting and quantifying enzymatic activity in samples. Its chromogenic properties make it suitable for colorimetric detection in diagnostic applications.

Additionally, used as a tracer in biochemical research to evaluate enzyme inhibitors or study conditions affecting enzyme activity, such as pH, temperature, and solvents. In some cases, it is employed in the synthesis of other organic compounds requiring a nitrophenyl group as an indicator or core structure.