Axon diameter varies up to 100-fold between distinct neurons in the central nervous system with larger axons exhibiting faster nerve conduction velocity. Further axon diameter influences myelination and its dynamic regulation might help fine-tune the timing of signal propagation and thus neural circuit function. Despite its importance, we know surprisingly little about how axons grow to such different diameters in the living nervous system and which mechanisms underlie this diversity. To fully understand how nervous systems are built, functionally mature, and remain healthy, we need to understand what regulates axon diameter growth. Thus, we developed a pipeline for automated high-resolution imaging of axonal diameters in zebrafish in vivo. We further established an automated image analysis pipeline which enabled a broad chemical-based screening approach for changes in axon diameter. This screen already identified novel compounds that impact the growth of myelinated axons in diameter. We can now investigate how these identified molecules influence axon diameter growth during development and over time by employing live imaging techniques in drug-treated and genetically modified zebrafish. The results will, in the future, support our understanding of how changes in axon diameter impact different parameters and the function of the myelinated axon unit.