Background & Purpose: Reactive oxygen species (ROS) play pivotal roles in cellular signaling. Nrf2 is a key transcription factor that regulates redox homeostasis. However, the relationship between Nrf2 and H2O2 is controversially discussed in the literature. Thus, we aimed to investigate how varying intracellular H2O2 levels, manipulated through different methods, influence Nrf2 regulation in brain endothelial cells under physiological normoxia (5 kPa O₂), contrasting with typical hyperoxic cell culture conditions. Experimental Approach: In this study, we developed and validated a novel oxygen-independent Nrf2 biosensor, Pericellular Oxygen-Insensitive Nrf2 Total LEvel Reporter (POINTER), using human cerebral microvascular endothelial cells (hCMEC/D3). We investigated the relationship between intracellular H2O2 and Nrf2 levels under varying oxygen conditions, employing exogenous H₂O₂ application, chemogenetic production of H2O2 via modified D-amino acid oxidase (mDAAO), and pharmacological induction of H2O2 with Auranofin. Key Results: POINTER confirmed significantly lower Nrf2 levels in hCMEC/D3 cells under physiological normoxia (5 kPa) compared to hyperoxia (room air). Auranofin demonstrated efficacy in modulating intracellular H2O2 increase comparable to exogenous administration of H₂O₂, yet H₂O₂ levels recovered to the baseline 24 hours after this treatment. Moreover, only chemogenetically produced H2O2 remained significantly elevated in cells after 24 hours. More importantly, only Auranofin, but not exogenous provision of H2O2 and chemogenetically induced oxidative stress robustly increased Nrf2 levels under physiological oxygen conditions demonstrated by the POINTER biosensor. Conclusion and Implications: These findings reveal the intricate regulation of Nrf2, indicating that both antioxidant inhibition and the elevation of H₂O₂ are crucial for modulating Nrf2 levels under physioxia.