Antigen-recognition by T cells requires the physical association with an antigen presenting cell (APC). At the interface between a T cell and an APC, the orchestrated redistribution of lipids, membrane receptors, and intracellular adaptors assembles a highly specialized junction, controlling the communication between the two cells, named the immunologic synapse (IS). The proper organization of the IS is a key step in host defense. Indeed, an appropriate T cell–APC interaction ensures the elimination of a wide range of pathogens and aberrant cells, whereas a deregulated IS formation leads to pathological situations such as infections, tumor development, or autoimmunity. Over the last decades T-cell scientists pioneered new imaging approaches to investigate IS assembly and organization. Microscopy techniques enable researchers to directly monitor, in space and time, the dynamics regulating T cell activation. In this chapter, we describe in detail different microscopy protocols to visualize and analyze the recruitment of different molecules and organelles at the IS.