The aim of this study was to address the urgent global need for an effective therapeutic strategy against acute stroke and perinatal asphyxia. A promising answer to limitations of currently existing therapies might be the newly designed Pathway Preferential Estrogen-1 (PaPE-1). Estrogen receptor-mediated signaling is pivotal for neuroprotection, and its impairment during hypoxia/ischemia may increase the risk of cerebral infarction. Our goal was to selectively activate non-nuclear estrogen receptors signaling, providing a wide therapeutic window and minimizing adverse hormonal effects associated with signaling through nuclear estrogen receptors. Our cellular model of post-treatment in perinatal asphyxia and stroke was constituted by primary neocortical cultures undergoing 6 hours of hypoxia and/or ischemia conditions followed by PaPE-1 being administered for subsequent 18 hours of reoxygenation period. The effectiveness of the treatment was validated by a variety of biochemical and molecular assays, e.g.: AlamarBlue viability assay, LDH release, Fluoro-Jade C staining, mitochondrial membrane potential measurements, neutral red uptake, ROS formation, and quantifying the expression of neurotoxicity- and apoptosis-related genes and proteins using qPCR and ELISAs. Our research proved that post-treatment with PaPE-1 protects brain neurons against hypoxia/ischemia by inhibiting neurotoxicity, neurodegeneration, along with enhancing cellular metabolic activity, reducing oxidative stress and expression of apoptosis-related factors such as FAS, FASL, BAX, BCL-2 and GSK3 β. These findings substantiated that targeting non-nuclear estrogen receptors with PaPE-1 serves as an effective therapeutic intervention against stroke and perinatal asphyxia. Funding: National Science Centre of Poland, grant number 2021/43/D/NZ7/00633.