Dementia, particularly Alzheimer's disease (AD), is a significant health challenge in Europe, characterized by tauopathy and the progressive loss of cholinergic neurons in the basal forebrain. Despite extensive research, effective treatments remain elusive. Utilizing neuroblastoma cells like SH-SY5Y offers a promising avenue to mimic neuronal phenotypes in vitro, aiding in disease mechanism exploration and therapy testing. However, establishing reliable differentiation protocols, especially under serum-free conditions, is imperative.This study aims to refine differentiation protocols and evaluate a serum-free neurobasal medium's efficacy in promoting SH-SY5Y neuronal differentiation, focusing on enhancing cholinergic phenotypes relevant to AD. Various iterations of a two-stage differentiation protocol were explored, all involving a 3-day treatment with 10 μM retinoic acid (RA) in the initial stage. Remarkably, only the variant incorporating 50 ng/ml brain-derived neurotrophic factor (BDNF) in the second stage significantly elevated neuronal markers in SH-SY5Y cells. Notably, this variant significantly elevated neuronal markers, including TAU isoforms and Microtubule-Associated Protein 2, indicating advanced neuronal maturation. Further, the preliminary data suggests the potential differentiation of parental SH-SY5Y cells into cholinergic neurons, with increased choline acetyltransferase levels exclusively in the BDNF-supplemented group. No significant changes were observed in other neurotransmitter markers, such as vesicular glutamate transporter 1 or tyrosine hydroxylase levels. These findings underscore the importance of optimized differentiation protocols and serum-free conditions in driving SH-SY5Y neuronal differentiation, offering insights into AD pathogenesis and potential therapeutic targets.