Receptor trafficking and signaling are intimately linked, especially in the Mu opioid receptor (MOR) where ligand-dependent endocytosis and recycling have been associated with opioid tolerance and dependence. Ligands of MOR can induce receptor endocytosis and recycling within minutes of exposure in heterologous systems and cultured neurons. Endocytosis removes desensitized receptors after their activation from the plasma membrane, while recycling promotes resensitization by delivering functional receptors to the cell surface. These rapid mechanisms can escape traditional analytical methods where only snapshots are obtained from highly dynamic events.Total internal reflection fluorescence (TIRF) microscopy is a powerful tool that can be used to investigate, in real time, surface trafficking events at the single molecule level. The restricted excitation of fluorophores located at or near the plasma membrane in combination with high sensitivity quantitative cameras makes it possible to record and analyze individual endocytic and recycling event in real time. In this chapter, we describe a TIRF microscopy protocol to investigate in real time, the ligand-dependent MOR trafficking in Human Embryonic Kidney 293 cells and dissociated striatal neuronal cultures. This approach can provide unique spatio-temporal resolution to understand the fundamental events controlling MOR trafficking at the plasma membrane.