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Phenotypic and functional assessment of Natural Killer (NK) cell activity against cancer cells in a clinical setting requires the integration of labor-intensive multicolor flow cytometry, chromium (Cr) release assays, and CD107a degranulation assays. To merge these into a single high-throughput assay, we developed a method for profiling NK-cancer cell interactions and simultaneous target killing using mass cytometry. In this study, a multiparameter 43-marker mass cytometry panel was applied to a co-culture of immune cells from healthy donors with diverse target cancer cell lines. DNA content, combined with classical CD45 surface staining, were used as gating parameters for co-cultures of immune cells (CD45high/DNAlow) with hematological (CD45low/DNAhigh) and solid cancer cell lines (CD45neg /DNAhigh), recapitulating flow cytometry forward and side scatter. This allows for discrimination of cancer cells from immune populations based on relative “size” and simultaneous assessment of phenotypical changes in both populations. In addition, the sensitivity of mass cytometry allows for the simultaneous detection of changes in NK cell phenotype and their degranulation upon target recognition, and for the analysis of target cells for cytotoxic protein granzyme B content, and cell death. These findings have broad applicability in clinical settings with the aim to phenotype and assess functional changes following NK-cancer cell interactions.