In this study, we established a drug screening system based on transcriptional regulation of vascular endothelial growth factor (VEGF) under hypoxia-inducible factor-1α control. We cloned the neomycin-resistance gene into the plasmid GL (pGL)3-promoter vector to generate the pGL3-promoter-neo vector. The 3 copies of the 47-bp fragment that contained the hypoxia response element of VEGF were synthesized and inserted in front of the minimal promoter of the pGL3-promoter-neo vector to generate p3HRE-luc-neo. The recombinant reporter gene vectors were transfected into EAhy926 cells, and stable cell lines were obtained. The positive cell line was selected for its ability to express luciferase in response to hypoxia. We demonstrated that CoCl₂ significantly enhances luciferase activity in a concentration-dependent fashion. We then optimized the cell density and incubation time under hypoxia which were used to screen. The assay exhibited a low background and was an ideal model for high-throughput screening for human VEGF regulators.