Widely used as a fluorogenic substrate in enzyme assays, particularly for detecting β-galactosidase activity. Upon enzymatic cleavage, it releases resorufin, a highly fluorescent compound, enabling sensitive and real-time monitoring of enzyme activity. Commonly applied in reporter gene assays, high-throughput screening, and cellular imaging due to its low background fluorescence and strong signal output. Also utilized in microbiology to assess bacterial load and gene expression in live cells. Its stability and sensitivity make it suitable for both in vitro and in vivo applications.