Axonal transport is key for the survival and function of all neurons. This process is especially important in basal forebrain cholinergic neurons due to their extremely long and diffuse axonal projections. These neurons are critical for learning and memory and degenerate rapidly in age-related neurodegenerative disorders like Alzheimer’s and Parkinson’s disease. The vulnerability of these neurons to age-related neurodegeneration may be partially attributed to their reliance on retrograde axonal transport for neurotrophic support. Unfortunately, little is known about the molecular biology underlying the retrograde transport dynamics of these neurons due to the difficulty associated with their maintenance in vitro. Here, we outline a protocol for culturing primary rodent basal forebrain cholinergic neurons in microfluidic chambers, devices designed specifically for the study of axonal transport in vitro. We outline protocols for labeling neurotrophins and tracking neurotrophin transport in these neurons. Our protocols can also be used to study axonal transport in other types of primary neurons such as cortical and hippocampal neurons.