NeuriteTracer is a novel, open-source ImageJ plugin developed for the automated quantification of neurite outgrowth. This macro analyzes fluorescence microscopy images of neurites and nuclei from dissociated cultured neurons. It accurately counts neuronal nuclei and measures neurite length based on user-defined thresholds. The tool has been validated for measuring neurite outgrowth from various neuron types, including cerebellar, dorsal root ganglion (DRG), and hippocampal neurons. Its measurements correlate strongly with those obtained from semi-manual tracing tools like NeuronJ and advanced analysis packages such as MetaXpress. NeuriteTracer is a valuable resource for in vitro assays focused on neuronal growth, development, and regeneration, capable of detecting subtle changes in neurite outgrowth in response to pharmacological treatments like the rho kinase inhibitor Y-27632. It offers a fast, simple-to-use, and fully automated solution for high-throughput screening of neuronal outgrowth assays, supporting 2D image dimensions.