During the natural killer (NK) cell immune response, cytoskeletal components, adhesion and signaling molecules and ligand-specific receptors are involved in the formation of the natural killer cell immune synapse (NK-IS), a highly organized supramolecular structure assembled at the interface of the interacting cells that regulates the NK cell activation and decides the target cell fate. In this review the current state on knowledge about the organization and physio-pathological function of the inhibitory, cytotoxic and activating NK-IS is presented. Moreover, it briefly summarizes microspectroscopy techniques suitable for live cell imaging of the dynamic biochemical processes, which achieve the coordinated NK cell immune responses.