Axonal transport is a process essential for neuronal function and survival that takes place on the cellular highways—the microtubules. It requires three major components: the microtubules that serve as tracks for the transport, the motor proteins that drive the movement, and the transported cargoes with their adaptor proteins. Axonal transport could be controlled by tubulin posttranslational modifications, which by decorating specific microtubule tracks could determine the specificity of cargo delivery inside neurons. However, it appears that the effects of tubulin modifications on transport can be rather subtle, and might thus be easily overlooked depending on which parameter of the transport process is analyzed. Here we propose an analysis paradigm that allows detecting rather subtle alterations in neuronal transport, as induced for instance by accumulation of posttranslational polyglutamylation. Analyzing mitochondria movements in axons, we found that neither the average speed nor the distance traveled were affected by hyperglutamylation, but we detected an about 50% reduction of the overall motility, suggesting that polyglutamylation controls the efficiency of mitochondria transport in axons. Our protocol can readily be expanded to the analysis of the impact of other tubulin modifications on the transport of a range of different neuronal cargoes.