CAR-T development necessitates the need for robust assays that are able to measure different critical quality attributes (CQAs) and can guide the development of these novel therapies from discovery to manufacturing. We have utilized real-time non-invasive impedance-based approach to develop potency assays for CD19 and EpCAM directed CAR-T cells. We can sensitively and quantitatively characterize the cytolytic activity of these engineered cells at different effector to target ratios under label-free conditions and in real-time. Additionally, using arginine, cytokine and serum preconditioning, we have shown that real-time potency assays can be used to optimize manufacturing conditions of engineered immune cells. Using oxygen consumption rate (OCR) and proton efflux rate (PER) we are able to show that pre-conditioning of engineered immune cells can modulate the spare respiratory capacity (SRC) of these immune cells which correlate with their potency and persistence. Additionally, using impedance and real-time imaging, we have developed an in vitro assay for migration/invasion of EpCAM directed CAR-T cells through the extracellular matrix coupled with selective killing of target tumor cells. Collectively, our data shows that real-time assessment of CQAs of engineered immune cells can provide incisive information for both discovery, development and manufacturing of these novel therapies.