Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with a unique or preferential expression on solid and liquid tumor cells in conjunction with a highly sensitive and real-time in vitro potency assessment that can inform the potency of the different CAR T cells. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols to generate CAR T cells for preclinical studies using lentiviral gene transduction, to expand CAR T cells, to validate CAR expression, and to utilize impedance-based real-time cellular analyzer monitoring to assess the potency of CAR T cells in vitro.