Free radicals are highly reactive compounds that lead to the onset of a variety of serious health conditions, known as “oxidative stress”. Antioxidants, on the other hand, act as defense mechanisms to fight the accumulation of free radicals and maintain cell homeostasis. Urtica dioica L. is a medicinal plant with unique antioxidant properties, mainly attributable to the presence of polar phenolic compounds. This study aimed to optimize the combination of determinant factors toward the maximum extraction of antioxidants from U. dioica L. Furthermore, it investigated the protective role of the extract on red blood cells that were exposed to oxidative stress. The extraction optimization was performed using Response Surface Methodology and the in vitro antioxidant activity of the extract was determined with Folin–Ciocalteu, FRAP, and DPPH assays. Based on the results, the highest value of antioxidant activity and polar phenolic compounds was recorded using 34% (v/v) ethanol as a solvent in an ultrasonic process carried out at 42 °C for 87 min. In addition, UV–Vis spectrum of the extract revealed the presence of chlorophylls, carotenoids, and flavonoid glycosides. This study also provided insight into the in vitro protective effect of the plant extract on red blood cells morphology under oxidative stress conditions. The findings highlighted the good predictability of the extraction model and the potential role of the extract as an antioxidant product.