The intracellular calcium flux assay is a widely used method for 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. 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 a high concentration. It has an 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.