In the past decade, a significant number of proteins involved in the developmental assembly and maturation of synapses have been identified. However, detailed knowledge of the molecular processes underlying developmental synapse assembly is still sparse. We have developed an approach that makes extended in vivo imaging of selected proteins in live Drosophila larvae feasible at a single-synapse resolution. This protocol describes the repetitive, noninvasive imaging of the neuromuscular junction (NMJ) of a live, intact, anesthetized Drosophila larva over extended periods of time with confocal microscopy. Proteins of interest must be tagged with a fluorescent label and have to be expressed in transgenic fly strains. The method has proven highly useful for the study of synaptic assembly and the trafficking of proteins. These data contribute to our understanding of synaptic assembly under in vivo conditions.