T cells play a critical role in maintaining the normal function of the adaptive immune response, with their dysfunction resulting in a variety of autoimmune and immunodeficiency diseases. Efficient and accurate detection of T cell function is therefore crucial to clinical diagnosis and development of immunomodulators. A variety of in vitro cellular systems are currently employed for analyzing T cell activation, yet all suffer from some combination of low throughput, unnatural conditions and long assay times. Label-free technologies are capable of detecting phenotypic responses to treatments under physiological conditions, thereby potentially accelerating drug discovery by facilitating the use of disease-relevant cell models for functional assessment and clinical diagnosis. The xCELLigence system is an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes in cells, such as those induced during T cell activation. Here we describe the development and validation of a T cell activation assay based upon electrical impedance. Co-activation of Jurkat cells with anti-CD28 and anti-CD3 functional antibodies led to impedance changes that were rapidly and sensitively recorded (within 30 minutes). This phenomenon was also observed in human peripheral blood mononuclear cells. These changes reflect morphological and adhesive alterations correlated with cytoskeletal reorganization as verified by microscopy. They were functionally dependent on canonical T cell signaling pathways, including calcium-mediated signals and Src family kinases because relevant inhibitors impaired T cell activation. Our results provide a convenient approach to measure T cell activation in real-time and to elucidate the underlying mechanisms of action through probing with small molecules.