In vitro models and cell lines were employed in this extensive investigation to explore the characteristics of the cold macerated methanol extract of Nerium oleander leaves (MENO), a traditionally used medicinal plant. Particular attention was paid to the plant's total flavonoid content, neuroprotective activity, and antioxidant effects. The findings provide insight into MENO's potential benefits and medical uses. The important metric known as total flavonoid content was measured precisely using a robust regression equation, demonstrating that MENO is a rich source of these potent antioxidants. Given that MENO showed concentration-dependent effect, it may be able to lessen oxidative stress and inflammation, two conditions that are linked to a variety of health problems. Its efficacy was further proven by IC50 values and comparisons with ascorbic acid. MENO was found to possess substantial antioxidant properties, as evidenced by its significant inhibition of lipid peroxide formation in both egg yolk and goat liver models, thereby protecting lipids and cellular membranes from oxidative damage. Interestingly, the study looked at the neuroprotective effects of MENO on neuroblastoma (SHSY-5Y) cells. It found dose-dependent effects and an IC50 value, indicating that MENO may be able to stop the proliferation of neuroblastoma (SHSY-5Y) cells. This study concludes by highlighting MENO's potential as a natural antioxidant source and potential neuroprotective agent. These findings provide a strong foundation for further investigation into the health benefits and potential applications of MENO, including in vivo studies. This research advances the fields of natural medicine and pharmaceutical sciences by providing new avenues for the development of medications and health-promoting chemicals.