Olfactory sensory neurons (OSN) regenerate from progenitors residing within the olfactory epithelium (OE). The OE pseudostratified architecture as well as soluble factors from the environment provide important cues for progenitors to differentiate into mature OSN. In case of severe injury with loss of cell-to-cell contact, regeneration is no longer possible: the OE is irreversibly replaced by respiratory, non-sensory epithelium. The molecular mechanisms triggering regeneration vs replacement of OSN are not fully understood. To address this, we developed a 2-D organotypic model which preserved the OE pseudostratified architecture. We investigated the influence of the coating ( laminin, fibronectin, or collagen IV). Survival and growth of OSN were further assessed comparatively in response to different growth factor treatments (BDNF, GDNF, NT3, retinoic acid, FGF, and combinations of those). To quantify axonal growth, an automated quantification tool was developed . Axonal growth was significantly increased in Fibronectin and Collagen IV conditions compared to Laminin and Control (p= 0.001). Furthermore, combinations of growth factors simultaneously inducing proliferation and differentiation were significantly more efficient compared to growth factors inducing only differentiation. In conclusion, the organotypic OE culture allows for efficient qualitative and quantitative assessment of OSN outgrowth in response to different coatings and growth factor treatments. The developed quantification tool was helpful to assess OSN outgrowth in an objective, accurate (ICC between automated and manual tracing: 0.96) and repeated manner, saving 87% of examination time. This model combined with the automated tracing tool provides a more representative and time-saving method to characterize OSN regeneration. It may facilitate high-throughput screens of regenerative compounds and transcription factors in the future. Funded by: Foundation Louis-Jeantet, Auris, and Sir Jules Thorn.