Due to the importance of CRAC (Orai1 + STIM1), Kv1.3 and KCa3.1 ion channels in the activation of T cells and proper operation of the immune system, the investigation of their topological relationship in the plasma membrane is an interesting study target and still an unexplored area of immunology. Those channels were shown to accumulate at the immunological synapse (IS) upon engagement to an antigen-presenting cell. Based on this, we hypothesized that Kv1.3 and Orai1 channels can make up signalosome when accumulated in the IS and localize in each other’s vicinity. To test this assumption, we studied the degree of colocalization of Kv1.3 and Orai1 upon the activation of the T cells, utilizing fluorescence microscopy and implemented an automated colocalization analysis pipeline. The results didn’t provide conclusive proof for the proposed hypothesis. We could detect medium to strong colocalization between Orai1 and Kv1.3 for control, CHO and Jurkat cells but not for cells in which we mimicked activation.