The function of neurons in afferent reception, integration, and generation of electrical activity relies on their strikingly polarized organization, characterized by distinct membrane domains. These domains have different compositions resulting from a combination of selective targeting and retention of membrane proteins. In neurons, most proteins are delivered from their site of synthesis in the soma to the axon via anterograde vesicular transport and undergo retrograde transport for redistribution and/or lysosomal degradation. A key question is whether proteins destined for the same domain are transported in separate vesicles for local assembly or whether these proteins are pre-assembled and co-transported in the same vesicles for delivery to their cognate domains. To assess the content of transport vesicles, one strategy relies on staining of sciatic nerves after ligation, which drives the accumulation of anterogradely and retrogradely transported vesicles on the proximal and distal side of the ligature, respectively. This approach may not permit confident assessment of the nature of the intracellular vesicles identified by staining, and analysis is limited to the availability of suitable antibodies. Here, we use dual color live imaging of proteins labeled with different fluorescent tags, visualizing anterograde and retrograde axonal transport of several proteins simultaneously. These proteins were expressed in rat dorsal root ganglion (DRG) neurons cultured alone or with Schwann cells under myelinating conditions to assess whether glial cells modify the patterns of axonal transport. Advantages of this protocol are the dynamic identification of transport vesicles and characterization of their content for various proteins that is not limited by available antibodies.