Cell therapy is poised to play a larger role in medicine, most notably for immuno-oncology. Despite the recent success of CAR-T therapeutics in the treatment of blood tumors and the rapid progress toward improved versions of both CAR- and TCR-Ts, important analytical aspects of preclinical development and manufacturing of engineered T cells remain immature. One limiting factor is the absence of robust multivariate assays to disentangle key parameters related to function of engineered effector cells, especially in the peptide-MHC (pMHC) target realm, the natural ligand for TCRs. Here we describe an imaging-based primary T cell assay that addresses several of these limitations. To our knowledge, this assay is the first quantitative, high-content assay that separates the key functional parameters of time- and antigen-dependent T cell proliferation from cytotoxicity. We show that the assay sheds light on relevant biology of CAR- and TCR-T cells, including response kinetics and the influence of effector:target ratio.