<p>Neurodegenerative diseases, such as Alzheimer’s disease (AD), are marked by gradual neuronal loss, frequently associated with mitochondrial dysfunction. Hentriacontane is a naturally occurring long-chain saturated hydrocarbon found in beeswax and certain herbal plants, and has antioxidant, anti-inflammatory and cytoprotective properties, suggesting its therapeutic&#xa0;potential in oxidative stress-associated neurodegeneration. Rotenone, a mitochondrial complex I inhibitor, induces oxidative stress and apoptosis, making it a widely used neurotoxic agent for investigating neurodegenerative disorders like AD. We initially performed in silico molecular docking, molecular dynamics, MM/GBSA, and ADMET pharmacokinetic analyses of hentriacontane against GSK3β to evaluate binding affinities and energies and assess the stability and dynamics of protein–ligand complexes. An in vitro drug safety assay was conducted using the MTT assay in SH-SY5Y cells. The cells were pretreated with 100&#xa0;μM rotenone two hours before the assay. A 96-well tissue culture-grade microplate was prepared with 100 μL of cell suspension (1 × 10⁴ cells/mL), and test compounds were added at concentrations of 20, 40, 60, 80, and 100&#xa0;μg/mL. Biochemical assays were conducted on cell supernatant to evaluate oxidative stress, proinflammatory, and apoptotic markers. Hentriacontane exhibited a significant binding affinity, with lower RMSD, RMSF, and a more negative ΔG_bind. ADMET analyses indicated favorable pharmacokinetic features. In vitro studies demonstrated that hentriacontane significantly enhanced cell viability, reduced oxidative stress markers, suppressed apoptotic and pro-inflammatory markers, and GSK3β protein levels. The collaborative significance of in silico and in vitro investigations suggests that hentriacontane may serve as a promising therapeutic candidate for mitigating oxidative stress-induced neurotoxicity via modulation of the GSK3β pathway.</p> Graphical Abstract <p></p>