T cell adhesion kinetics are a powerful indicator of target cell recognition during the cell-cell exploration process and formation of the immunological synapse facilitating cell communication and activation through specific intercellular molecular interactions. Various techniques have been used to document these binding kinetics, which foreshadow the dynamics of immunological synapse formation. Here, optical tweezers were used for studying at the level of single cells, the adhesion kinetics of human leukemia T lymphocyte cell line (CEM) to mouse mast cell line (P815) used as a tumor cell model. The P815 FcγRII receptors were saturated with the mouse anti-human CD3ɛ immunoglobulin G (OKT3) for initiating the T cell-P815 interaction through the engagement of the T cell CD3 nucleating the TCR complex formation structuring the synapse. Methods were developed to assess the time required to turn a contact between a T cell and a tumor cell into a stable interaction, and thus initiate the synapse formation. Single T cells were manipulated with the optical tweezers while the tumor cells were adhered to the glass surface under culture conditions. Three adhesions scenario were investigated by exerting either repetitive contacts engaging the same area of the two cells, repetitive contacts engaging the same area of the T cell but different areas on the tumor cell surface, or rolling the T cell over the tumor cell surface. With these methods, we observed that the median time of contact of CEM on P815 decreased in the presence of anti-CD3 OKT3 from 46 s to 1.3 s and the median rolling distance decreased from 50 μm to 1.8 μm prior the T cell immobilization. T cell adhesion speed assays can be used for measuring their lack of early response, identifying molecules involved in cell adhesion, or screening potential modulators. The techniques and quantitative methods, described here for studying T cell/target cell interaction based on manipulations using optical tweezers, can be generalized to all types of immunological or virological synapses as between T cell/dendritic cell, cytotoxic T cell/target, T cell/macrophage, T cell/B cell, NK cell/target, immune cell/infected cell and others.