Current protocols measure antibody-dependent cellular cytotoxicity (ADCC) in vitro using peripheral blood mononuclear cells (PBMCs), but isolation and variability among donors limit the viability and reproducibility of this approach. Here, we present a standardized co-culture model system to quantify ADCC on human breast cancer cells. We describe steps to engineer a natural killer cell line that stably expresses FCγRIIIa (CD16), required to mediate ADCC. We then detail the steps for the cancer-immune co-culture setup, followed by cytotoxicity measurement and analysis.Keywords: Antibody; Biotechnology and bioengineering; Cancer; Cell Biology; Cell culture; Cell isolation; Cell-based Assays; Flow Cytometry/Mass Cytometry; Immunology.