In vitro assays to measure neuronal growth are a fundamental tool used by many neurobiologists studying neuronal development and regeneration. The quantification of these assays requires accurate measurements of neurite length and neuronal cell numbers in neuronal cultures. Generally, these measurements are obtained through labor-intensive manual or semi-manual tracing of images. To automate these measurements, we have written NeuriteTracer, a neurite tracing plugin for the freely available image-processing program ImageJ. The plugin analyzes fluorescence microscopy images of neurites and nuclei of dissociated cultured neurons. Given user-defined thresholds, the plugin counts neuronal nuclei, and traces and measures neurite length. We find that NeuriteTracer accurately measures neurite outgrowth from cerebellar, DRG and hippocampal neurons. Values obtained by NeuriteTracer correlate strongly with those obtained by semi-manual tracing with NeuronJ and by using a sophisticated analysis package, MetaXpress. We reveal the utility of NeuriteTracer by demonstrating its ability to detect the neurite outgrowth promoting capacity of the rho kinase inhibitor Y-27632. Our plugin is an attractive alternative to existing tracing tools because it is fully automated and ready for use within a freely accessible imaging program.