6-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 an indicator of cell membrane integrity. The compound is non-fluorescent in its diacetate form but becomes highly fluorescent upon hydrolysis by intracellular esterases in living cells. This property makes it a valuable tool for distinguishing between live and dead cells in microscopy and flow cytometry.

Additionally, it is employed in tracking cell migration and proliferation in various research models. Its ability to permeate cell membranes and emit strong fluorescence upon activation allows for real-time monitoring of cellular processes. In drug discovery, it is used to assess the effects of compounds on cell health and viability. Furthermore, it finds applications in studying intracellular pH changes and enzyme activity, providing insights into cellular metabolism and function.