Neurofilaments are long flexible cytoplasmic protein polymers that are transported rapidly but intermittently along the axonal processes of nerve cells. Current methods for studying this movement involve manual tracking of fluorescently tagged neurofilament polymers in videos acquired by time-lapse fluorescence microscopy. Here, we describe an automated tracking method that uses particle filtering to implement a recursive Bayesian estimation of the filament location in successive frames of video sequences. To increase the efficiency of this approach, we take advantage of the fact that neurofilament movement is confined within the boundaries of the axon. We use piecewise cubic spline interpolation to model the path of the axon and then we use this model to limit both the orientation and location of the neurofilament in the particle tracking algorithm. Based on these two spatial constraints, we develop a prior dynamic state model that generates significantly fewer particles than generic particle filtering, and we select an adequate observation model to produce a robust tracking method. We demonstrate the efficacy and efficiency of our method by performing tracking experiments on real time-lapse image sequences of neurofilament movement, and we show that the method performs well compared to manual tracking by an experienced user. This spatially constrained particle filtering approach should also be applicable to the movement of other axonally transported cargoes.