Objectives: Over the past 50 years, retrospective epidemiological studies have consistently demonstrated an inverse association between the risk of developing Parkinson’s disease (PD) and history of tobacco use, with a two-fold risk decrease among tobacco smokers and snus users. In this study, we aimed to determine whether nicotine and other tobacco compounds could exert a neuroprotective role against alpha-Synuclein (α-Syn)-induced toxicity in a human dopaminergic neuronal model of PD. Methods: We differentiated human-induced pluripotent stem cells into dopaminergic neurons and then treated neuronal cultures with increasing doses of α-Syn preformed fibrils (PFF) or α-Syn monomers as controls. Various experimental conditions were tested using real-time and medium-throughput approaches: (i) neuronal adhesion/neurite outgrowth was assessed by impedance-based measurements using the xCELLigence® Real Time Cell Analyzer system, and (ii) mitochondrial respiration functions were evaluated using the Agilent Seahorse® analyzer. Results: Our results show that α-Syn PFF treatment induces dose-dependent neurodegeneration, neuritic pathology, and mitochondrial dysfunction in human dopaminergic neurons, thereby demonstrating the utility of this system to model molecular features of PD. Importantly, nicotine pretreatment for one hour was able to protect against multiple features of α-Syn PFF-induced toxicity in dopaminergic neurons in a dose-dependent manner. Current efforts are directed toward dissecting the mechanism through which nicotine exerts this observed beneficial effect. Conclusions: Our results provide a robust medium-throughput in vitro model of α-Syn PFF-induced toxicity in human dopaminergic neurons and pave the way toward a better understanding of mechanisms underlying the observed beneficial effects of nicotine in PD.