Antibody- and cell-based immunotherapies rely on the capacity of immune effectors to recognize and eliminate tumor cells. However, current in vitro cytotoxicity assays offer limited resolution, throughput, and mechanistic insight into which immune populations mediate killing. To address this gap, we developed a first-in-class, multiplexed mass cytometry platform that quantifies both effector-tumor binding and functional cytotoxicity at single-cell resolution across diverse immune conditions and therapeutic formats.