Neurons are highly polarized cells with their axon often extending over large distances away from the cell bodies (up to 1 meter in axonal length with a cell body less than 50 micrometer in diameter). Given that the majority of neuronal proteins and materials are synthesized in the cell body, such a long axon precludes effective diffusion of soma-produced proteins to their presynaptic destinations at the axonal terminals. As a result, neurons rely extensively on active axonal transport to deliver newly synthesized synaptic proteins, ion channels, lipids, and mitochondria to their axonal destinations via anterograde transport. On the other hand, retrograde axonal transport is responsible for carrying molecules and organelles destined for degradation from the axonal terminals back to the cell body. A highly efficient and tightly regulated machinery is thus required for a robust long-range transport of materials to ensure the neurons' proper growth, maintenance and survival. This thesis is a quantitative study of the underlying mechanism of axonal transport, with a specific focus on the retrograde axonal transport machinery in neurons. In our experimental setup, the axonal transport of cargos can be directly visualized in real-time using a neuronal microfluidic platform and fluorescence microscopy technique. We observe that cargos have high tendency to slow down their transport speed when crossing various obstacles along the axon such as non-moving cargos and stationary mitochondria. Single molecule study of retrograde nerve growth factor transport reveals that mechanical tug-of-war and intracellular motor regulation are complementary features of the near-unidirectional endosome directionality. Specifically, a stochastic mechanical simulation suggests that the endosomes are driven on average by 5-6 active dyneins and 1-2 down-regulated kinesins. This result is further supported by a study of the dynamics of endosomes detaching under load in axons, showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. Lastly, we present a quantitative characterization of the complex behavior of light-sensitive cryptochrome 2 (CRY2) protein under blue light. The results contribute to the understanding of the light-inducible CRY2 system and can be used as a guide to establish new optogenetic strategies to probe cellular processes in live cells.