The epidermal growth factor receptor (EGFR), a member of the erythroblastic leukemia viral oncogene homologue (ErbB) receptor tyrosine kinase family, plays key mitogenic signaling roles in development, cellular, and tissue physiology, as well as a myriad of malignancies. EGFR signaling occurs concurrently with ligand-receptor binding and subsequent endocytosis, and its signaling strength and engagement of different downstream signaling components are modulated by its endocytic trafficking itinerary. Understanding the factors and mechanisms that modulate ligand-bound EGFR's endocytic trafficking is therefore important for deciphering its role in pathophysiological processes. Endocytic trafficking of EGFR is regulated by a bunch of Rab small GTPases associated with the endocytic pathway. In this chapter, we describe a suite of relatively standard protocols in dissecting the role of a particular Rab protein in EGFR endocytic trafficking steps/stages. The approach constitutes a combination of genetic/molecular manipulations, followed by confocal imaging and a range of biochemical analyses. We shall mainly focus on Rab31 in our illustrations, but the approaches would be equally applicable to any Rab and its associated regulators/effectors.