Neuronal models of neurodegenerative diseases such as Parkinson’s Disease (PD) are extensively studied in pathological and therapeutical research with neurite outgrowth being a core feature. Screening of neurite outgrowth enables characterization of various stimuli and therapeutic effects after lesion. In this study, we describe an autonomous computational assay for a high throughput skeletonization approach allowing for quantification of neurite outgrowth in large data sets from fluorescence microscopic imaging. Development and validation of the assay was conducted with differentiated SH-SY5Y cells and primary mesencephalic dopaminergic neurons (MDN) treated with the neurotoxic lesioning compound Rotenone. Results of manual annotation using NeuronJ and automated data were shown to correlate strongly (R2-value 0.9077 for SH-SY5Y cells and R2-value 0.9297 for MDN). Pooled linear regressions of results from SH-SY5Y cell image data could be integrated into an equation formula (y=0.5410·x+1792; y=0.8789·x+0.09191 for normalized results) with y depicting automated and x depicting manual data. This automated neurite length algorithm constitutes a valuable tool for modelling of neurite outgrowth that can be easily applied to evaluate therapeutic compounds with high throughput approaches.