p-Nitrophenyl-β-D-galactopyranoside (pNPG) is widely used as a substrate in enzymatic assays to measure β-galactosidase activity. When β-galactosidase cleaves pNPG, it releases p-nitrophenol, a yellow-colored compound that can be easily quantified using spectrophotometry at 405 nm. This makes pNPG a valuable tool in molecular biology and biochemistry for studying enzyme kinetics, screening for enzyme inhibitors, and monitoring gene expression in systems like the lac operon. Additionally, it is employed in diagnostic tests to detect bacterial contamination, particularly in food and water samples, by identifying β-galactosidase-producing microorganisms. pNPG is also used to evaluate the ability of microorganisms to produce β-galactosidase, which aids in studying microbial efficiency in industrial processes such as dairy product manufacturing and wastewater treatment. Its high sensitivity and simplicity make it a preferred choice in both research and industrial applications.