One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor β (TGFβ), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.