NF-κB has been reported to play an important role in immunity, inflammation and carcinogenesis by regulating expression of its target genes. Thus, NF-κB was recognized as a promising target for the prevention and treatment drugs against cancer and inflammation. NF-κB sequence, reporter gene SEAP and selection gene for Zeocine resistance in plasmid pNiFty-SEAP were transfected into HEK293 cells. Therefore, NF-κB activity was able to be evaluated by SEAP activity in the transfected cells. Based on pNiFtySEAP/HEK293 cells, a screening assay was established, by which known NF-κB stimulators TNFα and PMA were demonstrated to activate the NF-κB in dose and time dependent manners. In contrast, NF-κB inhibitors PDTC and NAC showed inhibitory effects on the NF-κB activity in indicated doses and times. Except several characteristics such as stability, sensitivity, sepecifity, the assay was capable to screen both agents inducing and inhibiting NF-κB activity at the same experiment, which will be useful for the discovery of compounds targeting NF-κB signal pathway.