The intracellular calcium flux assay is a widely used method in monitoring signal transduction pathways and high throughput screening of G protein-coupled receptors (GPCRs) and calcium channel targets. Following the introduction of Fluo-3 in 1989, Fluo-4, Fluo-8, and Cal-520 were later developed with enhanced signal-to-background ratios and soon became the Ca2+ indicators of choice for confocal microscopy, flow cytometry, and high throughput screening applications. However, there are still a few caveats with Fluo-4. For example, like Fluo-3, Fluo-4 exhibits poor intracellular retention, and the use of probenecid is required to prevent the cell-loaded Fluo-4 from leaking out of cells. The use of probenecid with Fluo-4-based calcium assays compromises the assay results since probenecid is well-documented to have a variety of complicated cellular effects. Calbryte 520L, AM is a new fluorescent and cell-permeable calcium indicator. Like other dye AM cell loading, Calbryte 520L AM ester is non-fluorescent, and once it gets inside the cell, it is hydrolyzed by intracellular esterase and gets activated. The activated indicator is a polar molecule that is no longer capable of freely diffusing through the cell membrane and is essentially trapped inside cells. Calbryte 520L has a low affinity to calcium ions with a Kd ≤ 91 µM. Calbryte 520L produces a bright fluorescence signal in the presence of calcium at high concentrations. It has the identical excitation and emission wavelength as Fluo-4; thus, the same Fluo-4 assay settings can be readily applied to Calbryte 520L-based calcium assays.