G protein-coupled receptors (GPCRs) are cellular nanomachines that act as signaling hubs and account for ~40% of current drug targets. Endocytosis of GPCRs represents a major regulatory mechanism to sustain their downstream signaling within a stringent spatiotemporal regime. This is achieved by internalizing a fraction of the plasma membrane receptor population, thereby reducing the available pool of receptors for signaling at the cell membrane. In this article, we describe a detailed step-by-step hands-on guide that we have successfully used to monitor GPCR endocytosis and intracellular trafficking using serotonin1A receptor as a representative GPCR. For this, we carried out a judicious combination of flow cytometry and quantitative colocalization of confocal microscopic images to explore receptor endocytosis and trafficking. Notably, we applied this approach to demonstrate the role of membrane lipids (such as cholesterol) on endocytosis and trafficking of the serotonin1A receptor. Finally, we highlight important points and caveats to be considered, while carrying out endocytosis and trafficking experiments.