Understanding the immunological synapse formation and dynamics can be enriched by measuring cell-cell interaction forces and their kinetics. Microscopy imaging reveals structural organization of the synapse, while physical methods detail its mechanical construction. Various techniques have been reported or measuring forces needed to rupture the interface between a T lymphocyte and its target cell but most of them measure one pair at a time. We describe here a laminar shear flow-based method that exerts dragging forces on T cell-target cells pairs immobilized on the surface of a flow chamber. Increasing flow rate allows us to observe the detachment of hundreds of cell conjugates on the wide field of a light transmission microscope. Monitoring precisely the flow rate gradient exerted on T cells readily yields synapse rupture measurements. Dragging forces are measured at the point of rupture as a linear function of the flow speed in minutes from 10 pN to 20 nN for each cell pair among a statistically representative cell population in the whole field of view of a single experiment. The output cells can be collected in multi-well plate sorted in the increasing order of rupture forces. We used this approach to unveil the involvement of the cytoskeleton regulator adenomatous polyposis coli (APC) in the stability of immunological synapses formed between human cytotoxic T cell and tumor target cells. APC is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Reduced APC expression impairs T cell adhesion with tumor target cells suggesting an impact of APC mutation in anti-tumor immune defense.