The rapid increase of viral strains that are resistant to the currently available antiretroviral drugs is a threat to the success of current human immunodeficiency virus type 1 (HIV-1) treatment and emphasizes the importance of developing novel anti-HIV-1 compounds. To improve the current abilities to screen for novel HIV-1 inhibitors, here we introduce a T-cell-based reporter cell line (JLTRG-R5) that expresses both HIV-1 coreceptors, CXCR4 and CCR5, and provides the convenience of using enhanced green fluorescent protein (EGFP) as a direct and quantitative marker. Unlike previous EGFP-based reporter cell lines, JLTRG-R5 cells have an unusually high dynamic signal range, sufficient for plate reader detection using a 384-well format. In this format, JLTRG-R5 cell-based infectivity assays have a Z′-factor of 0.78, which defines the assay as extremely robust and clearly amenable to high-throughput screening. The functional similarity of the JLTRG-R5 cell line and peripheral blood mononuclear cells (PBMCs) was demonstrated through the identity of the inhibitory concentrations, 50% (IC50s) for four antiretroviral compounds or neutralizing antibodies. Because EGFP can be directly and continuously quantified in cell culture, the reporter cell line requires no manipulation during assay preparation or analysis. In addition, the EGFP marker allows for data acquisition at an optimal time point by prescreening selected positive control wells using fluorescent microscopy. These characteristics make the system extremely flexible, rapid, and inexpensive. Due to its intrinsic flexibility, the JLTRG-R5 cell-based reporter system provides a powerful tool to greatly facilitate future screening for HIV-1 inhibitors.