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. The intact larvae are anesthetized and noninvasively imaged with confocal microscopy. This method allows for both protein trafficking and protein turnover kinetics to be studied at various points in time during the development of an animal. These data contribute to our understanding of synaptic assembly under in vivo conditions. This protocol describes the assembly of the imaging chamber needed for in vivo imaging of Drosophila larvae. It also covers the construction of an appropriate anesthetization device.