Ischemic stroke is a disturbance in cerebral blood flow caused by brain tissue ischemia and hypoxia. We optimized a multifactorial in vitro model of acute ischemic stroke using rat primary neural cultures. This model was exploited to investigate the pro-viable activity of cell-penetrating peptides: arginine-rich Tat(49-57)-NH (RKKRRQRRR-amide) and its less basic analogue, PTD4 (YARAAARQARA-amide). Our model included glucose deprivation, oxidative stress, lactic acidosis, and excitotoxicity. Neurotoxicity of these peptides was excluded below a concentration of 50 μm, and PTD4-induced pro-survival was more pronounced. Circular dichroism spectroscopy and molecular dynamics (MD) calculations proved potential contribution of the peptide conformational properties to neuroprotection: in MD, Tat(49-57)-NH adopted a random coil and polyproline type II helical structure, whereas PTD4 adopted a helical structure. In an aqueous environment, the peptides mostly adopted a random coil conformation (PTD4) or a polyproline type II helical (Tat(49-57)-NH) structure. In 30% TFE, PTD4 showed a tendency to adopt a helical structure. Overall, the pro-viable activity of PTD4 was not correlated with the arginine content but rather with the peptide's ability to adopt a helical structure in the membrane-mimicking environment, which enhances its cell membrane permeability. PTD4 may act as a leader sequence in novel drugs for the treatment of acute ischemic stroke.