The study of dynamic cellular processes in living cells is central to biology and is particularly powerful when the motility characteristics of individual objects within cells can be determined and analysed statistically. However, commercial programs only offer a limited range of inflexible analysis modules and there are currently no open source programs for extensive analysis of particle motility. Here, we describe ParticleStats (http://www.ParticleStats.com), a web server and open source programs, which input the X,Y coordinate positions of objects in time, and output novel analyses, graphical plots and statistics for motile objects. ParticleStats comprises three separate analysis programs. First, ParticleStats:Directionality for the global analysis of polarity, for example microtubule plus end growth in Drosophila oocytes. Second, ParticleStats:Compare for the analysis of saltatory movement in terms of runs and pauses. This can be applied to chromosome segregation and molecular motor-based movements. Thirdly ParticleStats:Kymographs for the analysis of kymograph images, for example as applied to separation of chromosomes in mitosis. These analyses have provided key insights into molecular mechanisms that are not possible from qualitative analysis alone and are widely applicable to many other cell biology problems.