Fura-2 No Wash Calcium Assay Kit is a ratiometric calcium assay that allows homogeneous measurement of intracellular calcium mobilization caused by activation of G-protein-coupled receptors (GPCR) or calcium channels. Cells expressing a GPCR of interest that signals through calcium are pre-loaded with Fura-2 AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Fura-2 AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells and its fluorescence wavelength is blue-shifted upon binding to calcium. When cells are stimulated with agonists, the receptor signals the release of intracellular calcium, which greatly increase the fluorescence intensity of Fura-2 at the short wavelength. The characteristics of its ratio measurements reduce the effects of uneven dye loading and cell numbers, dye leakage and photo bleaching. The 340/380 nm excitation ratio allows accurate measurements of the intracellular Ca2+ concentration and makes this kit an ideal tool for more accurate measurement of cellular calcium concentration compared to Fluo-8 of the single wavelength. The kit provides an optimized assay method with no washing step for monitoring the G-protein-coupled receptors and calcium channels using HTS instrumentation. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format.