Cal-520 provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. Cal-520 AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520 AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal 520 AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520 AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520 calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists. Compared to other Cal-520 indicators,  Cal-520FF has the lowest affinity to calcium with Kd ~ 10 uM.