Seven transmembrane-spanning receptors, widely referred to as G protein-coupled receptors (GPCRs), mediate a broad spectrum of extracellular signals at the plasma membrane through G proteins, thereby modulating a variety of biological processes. In addition to G proteins, they also interact with a number of other cytoplasmic proteins. Thus, methods to understand GPCR signaling and their interactions with intracellular proteins in real time in live cells are of importance. Recent developments in microscopy methods and the availability of fluorescent proteins facilitated the development of techniques to unravel these interactions more precisely. This chapter describes the methodology for sequential capturing of images of membrane and cytoplasmic proteins fused to different fluorescence probes to understand GPCR interaction with cytosolic proteins and their colocalization.