Abstract: Immunotherapy for cancer aims to harness the immune system to target and eliminate tumors. Adoptive immunotherapy using immune cells engineered to express chimeric antigen receptors (CARs) has emerged as a promising therapy for B-cell malignancies. While CAR T and CAR NK cells are efficacious for certain patients, the effectiveness varies among individuals. It is therefore crucial to understand the factors responsible for disparate outcomes that determine treatment response so that biomarkers can be identified that predict the outcome of cell therapy. Cellular functional assays are particularly important in this context to provide a comprehensive understanding of immune cell behavior in vitro that will predict the function of the cells after infusion. To address the need for a comprehensive evaluation of immune cell function, we developed the Time-lapse Imaging Microscopy in Nanowell Grids (TIMING) platform. TIMING enables us to perform dynamic multi-parameter analysis at single-cell resolution to identify functional metrics associated with patients' responses. To accomplish this, we use the TIMING platform to acquire thousands of microscopy images of immune cells and tumor cells as they interact over time within micromachined nanowells. Our proprietary artificial intelligence (AI) algorithms then automatically identify and track single cells to quantify the kinetics of cell-cell interactions, immune cell health (migration, spontaneous death, and activated-induced cell death), potency (killing and serial killing), and cytokine release. By combining TIMING with an automated cell retrieval robot, single cells may be recovered for downstream analysis and identification of biomarkers associated with the observed cell behavior. In two independent studies, by profiling manufactured CAR T cell infusion products (IPs) of large B-cell lymphoma (LBCL) patients with the TIMING platform, we identified functional metrics (multifunctionality, high migration, and serial killing) and biomarkers (CAR T cell and tumor cell antigens) associated with CAR T cell anti-tumor activity. In summary, by applying multi-parameter analysis, the TIMING platform identified functional and molecular profiles associated with increased anti-tumor activity and better patient response. TIMING can be used to enhance the successful manufacturing of CAR T cells by identifying which cells perform best and why.