Cellular Ca2+ is essential to a large number of physiological processes ranging from embryonic development to muscle contraction. In silent status, the cytosol Ca2+ is maintained at a very low level around 100 nM, however, it would burst to several mM with appropriate stimulation. The calcium influx is triggered by activation of Ca2+ channels on the plasma membrane or release of Ca2+ from endoplasmic reticulum. Today, numerous Ca2+ indicators have been developed for quantification of this important second messenger, involving protein indicators such as aequorin and chemical indicators such as Fluo-4. Protein indicators stably expressed in the cell could significantly reduce screening complexity, thereby increasing throughput compared with chemical indicators which may need additional steps for dye loading. One drawback relates to the extra efforts required for generation of cell lines that express desired indicators: some of them are now commercially available (e.g., aequorin). In contrast, chemical Ca2+ indicators are more conveniently used nowadays due to their wide range of spectral properties, large detection windows and robust performance.