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 neuromuscular junctions (NMJs) are 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. Image analysis and quantification are best performed in three dimensions (3D; e.g., with the software Imaris by Bitplane), but they can also be performed using a simpler method for two-dimensional (2D) analysis using the free software ImageJ as presented in this protocol. We propose various possibilities for how an analysis may be performed with ImageJ, rather than providing an inflexible protocol, in which the steps must be followed without modifications. Although execution of most of the tools will be described via the ImageJ menu, most are also readily accessible through icons in ImageJ McMaster Biophotonics Facility (MBF) toolsets. These toolsets can also very easily be adapted for higher efficiency.