The extreme geometry of neurons spreads the need for mitochondrial functions out irregularly across vast cellular distances. This makes the long-distance transport of mitochondria a critical feature of their function in neurons. Axonal transport of mitochondria has been studied profitably in a variety of in vitro systems, particularly embryonic neurons grown in culture. This has allowed not only detailed motility analysis via light microscopy but also the ability to challenge the system with pharmacological agents and transfection. It does, however, carry caveats about its relevance to events in cells of the intact nervous system. In recent years, it has become possible to observe, quantify, and analyze the behavior of mitochondria within axons of the nervous system of live organisms. Here, we describe how to prepare the Drosophila larva for direct observation of mitochondrial axonal transport and how to gather and analyze motility data from this preparation, using confocal microscopy. This system takes advantage of our ability in Drosophila to express mitochondrially targeted fluorescent proteins in specific neuronal cell types, which allows us to visualize their traffic with ease, and to distinguish anterograde from retrograde traffic. Drosophila genetics also allows the analysis of mutations, gene overexpression, and knockdowns that affect mitochondrial function, including models of neurodegenerative disease. In addition, this preparation allows the visualization of the distribution and morphology of mitochondria in cell bodies within the central nervous system and in synapses. It is also possible to analyze mitochondrial functions other than transport, such as inner membrane potential, using this preparation.