Cellular processes depend on the fidelity of intracellular membrane traffic. Lipids, proteins, receptor ligands and solute molecules are trafficked to distinct compartments within the cell through both the biosynthetic and endocytic pathways. An appreciation of these pathways is vital for a complete understanding of intracellular drug delivery. Recent advances in fluorescence imaging have facilitated the analysis of these pathways in great detail. It is now possible to gain insight into the real-time dynamics of cellular components and macromolecular pharmacological agents as they are delivered into and traffic within single cells. Here, we discuss the analysis of intracellular drug delivery from the perspective of fluorescence imaging of both living and fixed cells. This review aims to cover trafficking pathways, markers for subcellular compartments, fluorescent labels for intracellular structures and pharmacological agents and relevant recent developments in imaging technology. In particular, we shall focus on the application of live cell imaging to the study of endocytic drug delivery.