The adult mammalian CNS is extremely limited in its ability to regenerate axons following injury. Glial scar, neuroinflammatory processes and molecules released from myelin impair axonal regrowth and contribute to the lack of neural regeneration. An in vitro assay that quantitates neurite outgrowth from cultured neurons as a model of neuronal regenerative potential is described. Specifically, the neurite outgrowth from primary neurons (rat cerebellar granule neurons; CGNs) and a neuronal cell line (NG108-15) were quantitatively measured after optimization of culture conditions. After cultures were fixed and immunostained to label neurons and nuclei, microscope images were captured and an image analysis algorithm was developed using Image-Pro Plus software to allow quantitative analysis. The algorithm allowed the determination of total neurite length, number of neurons, and number of neurons without neurites. The algorithm also allows for end-user control of thresholds for staining intensity and cell/nuclei size. This assay represents a useful tool for quantification of neurite outgrowth from a variety of neuronal sources with applications that include: (1) assessment of neurite outgrowth potential; (2) identification of molecules that can block or stimulate neurite outgrowth in conventional culture media; and (3) identification of agents that can overcome neurite outgrowth inhibition by inhibitory substrates.