Label-free approaches to assess cell properties ideally suit the requirements of cell-based therapeutics, since they permit to characterize cells with minimal perturbation and manipulation, at the benefit of sample recovery and re-employment for treatment. For this reason, label-free techniques would find sensible application in adoptive T cell-based immunotherapy. In this work, we describe the label-free and single-cell detection of in vitro activated T lymphocytes in flow through an electrical impedance-based setup. We describe a novel platform featuring 3D free-standing microelectrodes presenting passive upstream and downstream extensions and integrated into microfluidic channels. We employ such device to measure the impedance change associated with T cell activation at electrical frequencies maximizing the difference between non-activated and activated T cells. Finally, we harness the impedance signature of unstimulated T cells to set a boundary separating activated and non-activated clones, so to characterize the selectivity and specificity of the system. In conclusion, the strategy here proposed highlights the possible employment of impedance to assess T cell activation in label-free.