Aim:To develop homogeneous calcium mobilization assay for high-throughputscreening(HTS)of mas-related gene(Mrg)receptor agonists.Methods:CHO-Klcells stably expressing the full-length MrgD receptor and a calcium-sensitive dyewere used to develop an HTS assay based on intracellular calcium influx.Thismethod was applied to large-scale screening of a library containing 8000 syntheticcompounds and natural product extracts,cAMP measurements were carried outto verify the bioactivities of the hits found by the calcium mobilization assay.Similar approaches were also employed in the identification of the MrgA1 recep-tor agonists following HTS of 16000 samples.Results:EC_(50)values of the positivecontrol compounds(μ-alanine for MrgD receptor and dynorphin A for MrgA1receptor)determined by the calcium mobilization assay were consistent with thosereported in the literature,and the Z’factors were 0.65 and 0.50 for MrgD andMrgA1 receptor assay,respectively.About 31 compounds for the MrgD receptorand 48 compounds for the MrgA1 receptor showing20% of the maximal agonistactivities found in the controls were initially identified as hits.Secondary screen-ing confirmed that 2 compounds for each receptor possessed specific agonistactivities.Intracellular cAMP level measurements indicated that the 2 confirmedhits displayed the functionality of the MrgD receptor agonists.Conclusion:Aseries of validation studies demonstrated that the homogeneous calcium mobili-zation assay developed was highly efficient,amenable to automation and a robusttool to screen potential MrgD and MrgA1 receptor agonists.Its application maybe expanded to other G-protein coupled receptors that mobilize calcium influxupon activation. Aim: To develop homogeneous calcium mobilization assay for high-throughput screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-Klcells stably expressing the full- length MrgD receptor and a calcium-sensitive dyewere used to develop an HTS assay based on intracellular calcium influx. this method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts, cAMP measurements were carried out to verify the bioactivities of the hits found by the calcium mobilization assay. the identification of the MrgA1 recep-tor agonists following HTS of 16000 samples. Results: EC 50 values ​​of the positive control compounds (μ-alanine for MrgD receptor and dynorphin A for MrgA1 receptor) determined by the calcium mobilization assay were consistent with those reproduced the literature, and the Z factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptorand 48 compounds for the MrgA1 receptor showing 20% ​​of the maximal agonistactivities found in the controls were initially identified as hits. Secondary screen-ing confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. Conflusion: Aseries of validation studies demonstrated that the homogeneous calcium mobili-zation assay developed was highly efficient, amenable to automation and a robusttool to screen potential MrgD and MrgA1 receptor agonists. Use applications maybe expanded to other G-protein coupled receptors that mobilize calcium influxupon activation.