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We developed a comprehensive method for functional assessment of the changes in immune populations and killing activity of peripheral blood mononuclear cells (PBMCs) after cocultures with cancer cells using mass cytometry. In this study, a 43-marker mass cytometry panel was applied to a co-culture of immune cells from healthy donors' PBMCs with diverse cancer cell lines. DNA content, combined with classical CD45 surface staining, were used as gating parameters for co-cultures of immune cells (CD45/DNA) with hematological (CD45/DNA) and solid cancer cell lines (CD45/DNA). This strategy allows for universal discrimination of cancer cells from immune populations, without the need for a specific cancer cell marker and simultaneous assessment of phenotypical changes in both populations. The use of mass cytometry allows for the simultaneous detection of changes in natural killer (NK), NKT, and T cell phenotypes and the degranulation of immune populations upon target recognition, the analysis of target cells for cytotoxic protein granzyme B content, and cancer cell death. These findings have broad applicability in research and clinical settings with the aim to phenotype and assess functional changes following not only NK-cancer cell interactions, but also the effect of those interactions on other immune populations.