Several lines of evidence indicate that antibody-dependent cellular cytotoxicity (Wren et al., 2013) is important in the pathogenesis of HIV-1 infection. Namely, ADCC is induced during natural HIV-1 infection or in HIV-1 vaccine studies, the latter demonstrated by the RV144 vaccine trial. To expedite the assessment of ADCC in studies of HIV, we have developed a high throughput assay. We have optimized the rapid fluorometric antibody-mediated cytotoxicity assay (RFADCC) by transfecting the EGFP-CEM-NKr cell line to constitutively express SNAP-tagged CCR5. This cell line can then serve as a source of HIV-specific targets when coated with monomeric gp120, spinoculated with inactivated intact virions, infected by cell-free viral diffusion or infected by cell-to-cell transmission of virus. The optimized strategy has two significant advantages over the original RFADCC method: First, the preparation of detectable target cells is less labor intensive and faster as it does not rely on multiple staining and washing steps for target cells. Second, because the target cell markers GFP and SNAP are constitutively expressed, the assay provides highly reproducible data. These strengths make the optimized RFADCC assay suitable not only for studies of HIV-1 specific cytotoxicity but also for studies of cell-cell transmission of virus. In conclusion, this assay provides a new generation T cell line that can expedite large clinical studies as well as research studies in humans or non-human primates