Recent progress in biology and microscopy has made it possible to acquire multidimensional data on rapid cellular activities. Unfortunately, the data analysis needed to describe the observed biological process still remains a major bottleneck. We here present a novel method of studying membrane trafficking by monitoring vesicular structures moving along a three-dimensional cytoskeleton network. It allows the dynamics of such structures to be qualitatively and quantitatively investigated. Our tracking method uses kymogram analysis to extract the consistent part of the temporal information and to allow the meaningful representation of vesicle dynamics. A fully automatic extension of this method, together with a statistical tool for dynamic comparisons, allows the precise analysis and comparison of overall speed distributions and directions. It can handle typical complex situations, such as a dense set of vesicles moving at various velocities, fusing and dissociating with each other or with other cell compartments. The overall approach has been characterized and validated on synthetic data. We chose the Rab6A protein, a GTPase involved in the regulation of intracellular membrane trafficking, as a molecular model. The application of our method to GFP-Rab6A stable cells acquired using fast four-dimensional deconvolution video-microscopy gives considerable cellular dynamic information unreachable using other techniques.