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Background This study aimed to establish an in vitro model of Alzheimer’s Disease (AD) to investigate the neuroprotective activities of allyl isothiocyanate ( AITC) and sulforaphane ( SFN). Materials and Methods Human neuroblastoma cell lines (SKNAS) were used for the in vitro model of AD after amyloid-β25−35 (Aβ25–35) treatment. Cytotoxicity analysis was performed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Indirect immunocytochemical methods were used to assess the tau protein, alpha-synuclein (α-synuclein), and β-amyloid distribution in the in vitro model of AD and SKNAS cells. Results An in vitro AD model was induced by treatment of SKNAS cells with 1 µM of Aβ25–35 for 48 h. AITC and SFN were applied for 48 h, and the optimal concentrations were determined as 50 µM AITC and 15 µM SFN. Reduced tau immunoreactivity was shown after AITC and SFN administration in SKNAS cells and in vitro models, demonstrating that AITC and SFN prevented amyloid plaque production in the in vitro AD model control group by reducing the β-amyloid level, α-synuclein levels were similar in control and in vitro AD model cells. Reduced α-synuclein levels were observed after SFN treatment in the AD model cells and AITC treatment in the control cells. Conclusion It could be concluded that AITC and SFN are potential components as neuroprotective agents against AD.