Axonal transport is crucial for neuronal homeostasis, survival, and development. Indeed, axonal transport needs to be precisely regulated for developing axons to swiftly and accurately respond to their complex and evolving environment in space and time. A growing number of studies have started to unravel the diversity of regulatory and adaptor proteins required to orchestrate the axonal transport machinery. Despite some discrepancies between in vitro and in vivo axonal transport studies, most analyses aiming at deciphering these regulatory complexes, as well as their mode of action, were carried out in vitro in primary cultures of neurons, and mainly focused on their impact on axon specification and elongation, but rarely on axon navigation per se. Given the clear influence of the in vivo environment on axonal transport, including chemical and physical interactions with neighboring cells, it is essential to develop in vivo models to identify and characterize the molecular complexes involved in this key process. Here, we describe an experimental system to monitor axonal transport in vivo in developing axons of live zebrafish embryos with high spatial and temporal resolution. Due to its optical transparency and easy genetic manipulation, the zebrafish embryo is ideally suited to study such cellular dynamics at a single axon scale. Using this approach, we were able to unravel the key role of Fidgetin-like 1 in the regulation of bidirectional axonal transport required for motor axon targeting. Moreover, this protocol can be easily adapted to characterize a wide range of axonal transport regulators and components in physiological conditions and may additionally be used to screen new therapeutic compounds based on their ability to recue axonal transport defects in pathological conditions.