5-Carboxyfluorescein diacetate is widely used as a fluorescent tracer in biological and cellular studies. Its primary application is in cell viability assays, where it serves as a non-toxic, cell-permeable dye. Once inside live cells, esterases cleave the diacetate groups, converting it into a fluorescent compound that is retained within cells with intact membranes. This property makes it a valuable tool for distinguishing between live and dead cells in microscopy and flow cytometry. Additionally, it is employed in cell tracking and migration studies, as well as in monitoring cell proliferation and cytotoxicity in various research settings. Its ability to emit strong fluorescence under specific wavelengths enhances its utility in real-time imaging and quantitative analysis.