Abstract: Glucagon-like peptide-1 (GLP-1) could increase the level of cyclic adenosine monophosphate (cAMP) in cells to stimulate insulin secretion in β cells of pancreas. So GLP-1 analogues, such as liraglutide, have become new anti-hyperglycemia drugs for type 2 diabetes. In this study, a set of in vitro activity detection method suitable for GLP-1 analogues was established using GLP-1R-GFP (green fluorescent protein, GFP)-HEK293A cells which stably expressing GLP-1 receptor (GLP-1R). After optimizing the detection parameters such as assay sensitivity, cell density, and the incubation condition, the cAMP content level of GLP-1R-GFP-HEK293A cells stimulated by four GLP-1 analogues, such as liraglutide, were detected by homogeneous time-resolved fluorescence (HTRF). The values of concentration for 50% of maximal effect (EC₅₀) of GLP-1 analogues were calculated by cAMP dose-response curve to evaluate the in vitro activity of those drugs. In addition, enzyme-linked immunosorbent assay and quantitative polymerase chain reaction were applied to determine the content of host cell protein and host residual DNA, respectively. This study provides a stable, reliable, and sensitive in vitro activity analysis and host impurity detection method for high-throughput screening of GLP-1 analogues.