The 2010 UNAIDS report states that approximately 34 million people are living with human immunodeficiency virus type 1 (HIV-1), despite highly active antiretroviral therapy (HAART). Despite being effective, ARV therapy has many disadvantages including a cost trajectory unsustainable for economically challenged countries, serious side effects, and the development of drug-resistant strains. Several measures are under way to develop alternatives for ARV therapy, particularly for the control of early HIV-1 infection, but lack of efficient drug targets and assays hinders the search of potential ARV molecules. The dendritic cells present in the mucosal tissue, together with CD4+ T lymphocytes and macrophages, are among the first cells to encounter HIV-1. The dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) molecule plays a crucial role in binding HIV-1 through high affinity interaction with viral envelope glycoprotein gp120. DC-SIGN, a mannose-binding C-type lectin expressed on cells in the mucosal tissue of the rectum, uterus and cervix, facilitates early HIV-1 infection after sexual transmission. In this study we report a novel target-specific high-throughput screening (HTS) assay capable of quantifying the binding as well as the inhibition of DC-SIGN and gp120. The specificity of the assay was determined through competitive inhibition while optimization occurred for DMSO tolerance (0.5%), Z’ factor (0.51), signal-to-noise ratio (3.26), and coefficient of variation (5.1%). For assay validation previously recognized antagonists of DC-SIGN/gp120 binding were tested to detect inhibition demonstrating the suitability of the assay for future HTS screen of potential inhibitors that block the binding between DC-SIGN and gp120 which may prevent early HIV-1 infection.