Specific interactions of the human hepatitis B virus (HBV) surface proteins with the core protein of nucleocapsid are critical for the envelopment of virus particles, and inhibition of this process may prevent the production of infectious virus. A modified enzyme-linked immunosorbent assay (ELISA), which measured the interaction between the core protein and PreS region of the surface protein, was used to screen a chemical library for compounds that would block this interaction. Few inhibitory compounds were identified from a chemical library consisting of 5600 compounds. Among them, two compounds inhibited the production of HBV particles from transiently HBV-producing HuH7 cells. The IC50 values of these compounds for inhibition of HBV production in HuH7 cells were in the micromolar concentration range. These results indicate that compounds that prevent the interaction between the core protein and PreS region of the surface protein may possibly be useful as anti-HBV agents.