The use of primary neuronal cultures generated from Drosophila tissue provides a powerful model for studies of transport mechanisms. Cultured fly neurons provide similarly detailed subcellular resolution and applicability of pharmacology or fluorescent dyes as mammalian primary neurons. As an experimental advantage for the mechanistic dissection of transport, fly primary neurons can be combined with the fast and highly efficient combinatorial genetics of Drosophila, and genetic tools for the manipulation of virtually every fly gene are readily available. This strategy can be performed in parallel to in vivo transport studies to address relevance of any findings. Here we will describe the generation of primary neuronal cultures from Drosophila embryos and larvae, the use of external fluorescent dyes and genetic tools to label cargo, and the key strategies for live imaging and subsequent analysis.