In order to find agonists for melanocortin 4 receptor (MC4R), an assay in vitro was established for high throughout screening. The MC4R receptor plasmid (MC4R/pCDNA3.1) and reporter gene plasmid (3×CRE/3×MRE/SRE-LUC) were cotransfected HEK293 to establish a cell line for agonist screening. To identify and optimize the assay condition, the effects of some factors were examined by using α-MSH, such as cell number per well, incubation time, final concentration of DMSO and luciferase's substrate concentration on the assay. A steady cell line and a reliable method were established for MC4R agonist screening, the Z'-factor value was near to 0.7 on the condition in each well that the cell number were 4×10⁴ , the agonist incubation time was 8 h, the final concentration of DMSO was less than 1%, and the final concentration of α-MSH was 1 μmol/L. This technology can be applied to identify agonist for MC4R receptor.