T-cell exhaustion is characterized by low proliferative capacity, suppressed effector function, and the persistent expression of co-suppressive receptors such as PD-1 and TIM-3. Chronic activation of T cells, as seen in chronic viral infections or cancer, leads to T-cell exhaustion. In addition, T-cell exhaustion is a major obstacle to the clinical application of T-cell-dependent bispecific antibodies (TDBs). On the other hand, blocking the co-suppressive receptors has improved T-cell function, highlighting the importance of recovery from T-cell exhaustion to enhance therapeutic efficacy. Ongoing research focuses on recovering from exhausted T cells using genetic engineering, particularly in CAR-T cell therapy. Although mouse models infected with LCMV are widely used to generate exhausted T cells, this method raises concerns about animal welfare. We established a method using magnetic beads to induce T-cell exhaustion in vitro, simulating the immune state caused by chronic activation. Moreover, this in vitro exhaustion-induction method is applicable to the evaluation of TDB functional activity and the examination of the T-cell manipulation methods. Collectively, the current in vitro T-cell exhaustion assay system will be helpful for the development of TDBs to overcome T-cell exhaustion.