Abstract Aim: To develop a universal high-throughput screening assay based on Gα15/16-mediated calcium mobilization for the identification of novel modulators of G-protein-coupled receptors (GPCR). Methods: In the present study, CHO-K1 or HEK293 cells were co-transfected with plasmids encoding promiscuous G-protein Ga15/16 and various receptors originally coupled to Gαs, Gαi, or Gαq pathways. Intracellular calcium change was monitored with fluorescent dye Fluo-4. Results: We found out for all the receptors tested, Gα15/16 could shift the receptors’coupling to the calcium mobilization pathway, and the EC50 values of the ligands generated with this method were comparable with reported values that were obtained using traditional methods. This assay was validated and optimized with the -opioid receptor, which originally coupled to Gαi and was recently found to play important roles in neurodegenerative and autoimmune diseases. A large-scale screening of 48 000 compounds was performed based on this system. Several new modulators were identified and confirmed with the traditional GTPS binding assay. Conclusion: This cell-based calcium assay was proved to be robust and easy to automate, and could be used as a universal method in searching for GPCR modulators.