Used as a substrate in enzymatic assays, particularly for alkaline phosphatase activity, enabling the detection and quantification of enzymes in biochemical research. Upon hydrolysis by the enzyme, it releases 4-nitrophenol, which produces a yellow color detectable by spectrophotometry at 405 nm. It is also employed in diagnostic kits for medical testing, where it helps measure enzyme levels in clinical samples. In molecular biology, it serves as a chromogenic agent, producing a detectable color change upon enzymatic hydrolysis, aiding in visualization and analysis. Additionally, it finds use in environmental monitoring to assess phosphatase activity in soil and water samples, providing insights into microbial activity and nutrient cycling.