Neurotrophic activity constitutes a crucial factor in the recovery from neurological injuries and is impaired in neurodegenerative disorders. Preclinical studies of neurotrophic factors to improve outcome of neurodegenerative diseases have yielded promising results. However, due to the complexity of these therapies, the clinical translation of this approach was so far not successful and more feasible treatments with neurotrophic activity may be promising alternatives. Therefore, highly sensitive and robust assays for compound screening are required. Nerve growth factor (NGF) is known to induce Neurofilament-L (NF-L) expression in a rat pheochromocytoma cell line (PC12) during early neuronal differentiation. We generated and characterized an enhanced green fluorescent protein (EGFP)-NF-L reporter PC12 cell line for the development of a cell-based assay that allows straightforward quantification of early neuronal differentiation based on NF-L expression. Using Cerebrolysin®, a neuropeptide preparation that mimics the action of endogenous neurotrophic factors, as a role model for a compound that stimulates neurotrophic activity in the central nervous system, this cell-based assay was proved to be a robust, specific, and reproducible method. The EGFP-NF-L reporter PC12 cell line holds promise for more efficient and reliable compound testing for assessing neurotrophic activity. Moreover, the straightforward approach of evaluating the EGFP reporter makes it a useful model system for studying NF-L signaling.