Used primarily as a substrate in biochemistry and molecular biology to detect the presence of β-galactosidase enzyme activity. When the enzyme cleaves the glycosidic bond, it releases o-nitrophenol, which turns yellow and can be measured spectrophotometrically at 420 nm. This makes it a valuable tool in assays for gene expression studies, particularly in lacZ reporter systems. It is also employed in microbiology to identify and differentiate bacterial species based on their ability to produce β-galactosidase. Additionally, it aids in studying enzyme kinetics and inhibition mechanisms.