We describe a strategy for fluorescent imaging of organelle transport in primary hippocampal neurons treated with amyloid-β (Aβ) peptides that cause Alzheimer's disease (AD). This method enables careful, rigorous analyses of axonal transport defects, which are implicated in AD and other neurodegenerative diseases. Moreover, we present and emphasize guidelines for investigating Aβ-induced mechanisms of axonal transport disruption in the absence of nonspecific, irreversible cellular toxicity. This approach should be accessible to most laboratories equipped with cell culture facilities and a standard fluorescent microscope and may be adapted to other cell types.