Used as a sensitive substrate in enzyme assays, particularly for detecting alkaline phosphatase activity. When dephosphorylated by the enzyme, it yields fluorescein, a highly fluorescent compound that can be easily quantified using fluorometry. This reaction is widely applied in immunoassays, such as ELISA, and in molecular biology for nucleic acid detection, where signal amplification through fluorescence allows for high detection sensitivity. It is also employed in histochemistry to visualize enzyme distribution in tissues and cells. Due to its low background fluorescence in the phosphorylated form, it provides excellent signal-to-noise ratios in diagnostic and research applications.