Hepatitis B is an infection of the liver caused by the hepatitis B virus (HBV) in which transmission can occur vertically from infected mother to child or horizontally by blood, blood products and sexual transmission. Approximately 350 million people are estimated to be chronically infected with HBV and a million people die each year from HBV-related chronic liver disease, including cirrhosis and hepatocellular carcinoma, in the world. Currently, nucleoside and nucleotide analogues (lamivudine, adefovir, entecavir, tenofovir, etc.) are widely used for treatment of chronic HBV infection. However, current therapies do not eradicate HBV infection. The persistence of HBV covalently closed circular DNA (cccDNA), which serves as a template for viral replication, is a substantial risk of HBV reactivation. Furthermore, long-term treatment with nucleoside/nucleotide analogues frequently results in the emergence of drug-resistant HBV. Thus, the development of a new anti-HBV drug with a different mode of action is expected. In order to develop novel anti-Hepatitis B virus (HBV) agents, we developed two distinct cell- and plasmid-based assays targeting the HBV replication machinery, i.e. (1) HBV replication assay, a plasmid-based measurement of the HBV reverse-transcription by real-time PCR; (2) HBc-HBc assay, a method that can quantify the protein-protein interacion of HBV core protein (HBc) by utilizing bimolecular luminescence complementation of split-luciferase. By screening about 140,000 compounds with efficient combination of two assays, we successfully obtained hit compounds that selectively inhibit HBV replication. The two assays we have developed are suitable for the discovery and development of novel anti-HBV agents.