Current live-cell imaging techniques make possible the observation of live events and the acquisition of large datasets to characterize the different parameters of the visualized events. They provide new insights into the dynamics of biological processes with unprecedented spatial and temporal resolutions. Here we describe the implementation and application of a new tool called TrackAnalyzer, accessible from Fiji and ImageJ. Our tool allows running semi-automated single-particle tracking (SPT) and subsequent motion classification, as well as quantitative analysis of diffusion and intensity for selected tracks relying on the graphical user interface (GUI) for large sets of temporal images (X–Y–T or X–Y–C–T dimensions). TrackAnalyzer also allows 3D visualization of the results as overlays of either spots, cells or end-tracks over time, along with corresponding feature extraction and further classification according to user criteria. Our analysis workflow automates the following steps: (1) spot or cell detection and filtering, (2) construction of tracks, (3) track classification and analysis (diffusion and chemotaxis), and (4) detailed analysis and visualization of all the outputs along the pipeline. All these analyses are automated and can be run in batch mode for a set of similar acquisitions.