Drosophila is an established system in which to study synaptic development, function, and plasticity. A particular advantage of the larval neuromuscular system is its consistent well-defined segmental arrangement of neurons and muscle targets. Indeed, the motor neurons of the Drosophila central nervous system are particularly well characterized in terms of origin, identity, morphology, and electrophysiology, and have been used for studies on axonal transport of organelles, vesicle trafficking, and recycling. To facilitate the identification of nerves and synapses in vivo, specific fluorescent protein markers can be used. For example, UASmCD8-green fluorescent protein (GFP) and UASmyr-red fluorescent protein (RFP) both preferentially label plasma membranes, whereas Discs large (DLG) reveals synapses. Gal4 drivers can be used to target all neurons (e.g., elavGal4) or specifically label motor neurons (e.g., d42Gal4). This protocol describes the dissection of Drosophila larvae to isolate neurons for live cell imaging.