Assembly of human immunodeficiency virus (HIV-1) represents an attractive target for antiretroviral therapy, which is not exploited by currently available drugs. We established high-throughput screening for assembly inhibitors, based on competition of small molecules for the binding of a known dodecapeptide assembly inhibitor to the C-terminal domain of HIV-1 CA (capsid). Screening of >70,000 compounds from different libraries identified 2-arylquinazolines as low micromolecular inhibitors of HIV-1 capsid assembly. We prepared focused libraries of modified 2-arylquinazolines and tested their capacity to bind HIV-1 CA, to compete with the known peptide inhibitor, and to prevent the replication of HIV-1 in tissue culture. Some of the compounds showed potent binding to the C-terminal domain of CA, and were found to block viral replication at low micromolar concentrations.