Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid (βAP) protein in the brain, which is associated with neurodegeneration. This study investigates the relationship between βAP aggregation and its neurotoxic properties in vitro. βAP peptides were synthesized and tested for aggregation and toxicity. Results showed that βAPs containing the hydrophobic β29–35 region formed stable aggregates over time, and these aggregates were associated with neurotoxicity. In contrast, a synthetic peptide from islet amyloid polypeptide (IAP) aggregated but did not exhibit toxicity, suggesting that βAP-induced neurotoxicity is specific to βAP. Neurotoxicity was observed in short-term neuronal cultures but not in astrocyte cultures, supporting the hypothesis that βAP contributes to AD neurodegeneration. Aggregation of βAP was confirmed using light microscopy, SDS-PAGE, and centrifugation assays. Aggregated βAPs caused significant neuronal degeneration, while non-aggregated βAPs did not. Reversing βAP aggregation by resolubilization in HFP reduced toxicity, indicating that aggregation is a key factor in βAP neurotoxicity. The study also found that βAP-induced neurotoxicity in long-term cultures was not prevented by MK801, a glutamate antagonist, suggesting that the mechanism is not related to glutamate receptor activation. Overall, the data support the hypothesis that βAP aggregation is a critical factor in βAP-induced neurotoxicity and contributes to AD neurodegeneration.Alzheimer's disease (AD) is characterized by the accumulation of β-amyloid (βAP) protein in the brain, which is associated with neurodegeneration. This study investigates the relationship between βAP aggregation and its neurotoxic properties in vitro. βAP peptides were synthesized and tested for aggregation and toxicity. Results showed that βAPs containing the hydrophobic β29–35 region formed stable aggregates over time, and these aggregates were associated with neurotoxicity. In contrast, a synthetic peptide from islet amyloid polypeptide (IAP) aggregated but did not exhibit toxicity, suggesting that βAP-induced neurotoxicity is specific to βAP. Neurotoxicity was observed in short-term neuronal cultures but not in astrocyte cultures, supporting the hypothesis that βAP contributes to AD neurodegeneration. Aggregation of βAP was confirmed using light microscopy, SDS-PAGE, and centrifugation assays. Aggregated βAPs caused significant neuronal degeneration, while non-aggregated βAPs did not. Reversing βAP aggregation by resolubilization in HFP reduced toxicity, indicating that aggregation is a key factor in βAP neurotoxicity. The study also found that βAP-induced neurotoxicity in long-term cultures was not prevented by MK801, a glutamate antagonist, suggesting that the mechanism is not related to glutamate receptor activation. Overall, the data support the hypothesis that βAP aggregation is a critical factor in βAP-induced neurotoxicity and contributes to AD neurodegeneration.