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Summary: Here, we present a comprehensive protocol for the generation and functional characterization of chimeric antigen receptor (CAR) T cells and their products by mass cytometry in a reproducible and scalable manner. We describe the production of CAR T cells from human peripheral blood mononuclear cells. We then detail a three-step staining protocol with metal-labeled antibodies and the subsequent mass cytometry analysis. This protocol allows simultaneous characterization of CAR T cell intracellular signaling, activation, proliferation, cytokine production, and phenotype in a single assay.