Single-molecule localization microscopy (SMLM) has provided unique insight into the biophysical and nanoscale molecular mechanisms that underpin fundamental cellular functions. Nonetheless, due to technical limitations and challenges, much of the current work on real-time SMLM has relied heavily on in vitro or in cellulo model systems, which may not comprehensively reflect molecular dynamics in vivo. The current chapter acts as a guide for experimenters aiming to extend their research into in vivo or ex vivo SMLM, highlighting the obstacles and challenges that become apparent when attempting to elucidate the nanoscale cellular environment within complex multicellular organisms. Here, we describe our procedure for performing in vivo SMLM at the transgenic Drosophila melanogaster larval neuromuscular junction and provide insight into how to overcome these challenges. The protocol described allows direct visualization of molecules involved in synaptic communication within in vivo presynaptic nerve terminals and can be extended to other live multicellular organisms and systems.