Proteins on the two-dimensional surface of cell membrane continuously move. However, it is still unidentified how proteins move on the cell surface and what is the role of protein’s diffusivity on cell’s life. Particularly dynamic interactions between membrane proteins determine correct cellular responses to diverse environmental changes. Because various protein complexes on a plasma membrane participate in cellular functions, technique identifying complex interactions of membrane proteins with spatio-temporal resolution in a single living cell is highly demanding. Real-time tracking, i.e., measuring the trajectories of individual proteins, can isolate such complex interactions in living cells. In this talk, I will introduce the basic concept of the novel biophysical approach of single-particle tracking, and then present how to image membrane protein interactions using the accurate measurement of diffusion coefficient of membrane proteins in a single living cell. We validated that our approach can measure the interactions between ligands and membrane proteins using an epidermal growth factor receptor and show its applicability in measuring dissociation constant of ligands and subpopulation dynamics in a single living cell. We extended our technique to G protein-coupled receptors, which have been the targets of 40% of the modern drugs. Our results demonstrate that single-particle tracking would be a powerful technique for investigating the various interactions of membrane proteins and the processes of signal transduction in a single living cell.