The most common and widely transplanted tissue worldwide is blood. But concerns about safety and adequacy of blood transfusion have fostered 20 years of research into blood substitutes such as oxygen carriers based on modified hemoglobin (Hb). Chemically modified or genetically engineered Hb developed as oxygen therapeutics are designed to restore blood volume and to correct oxygen deficit due to ischemia in a variety of clinical settings. Uncontrolled oxidative reactions mediated by large amounts of cell-free Hb and their reactions with various oxidant/antioxidant and cell signalling systems emerge as an important pathway of toxicity. Hemoglobin can react with oxygen and NO, leading to the production of reactive oxygen or nitrogen species. Inside the bloodstream, oxidized Hb and ROS/RNS are in direct contact with endothelial cells (EC). Thus, chain reactions may trigger molecular and cellular biology, causing oxidative stress-related pathologies. This editorial presents an overview of interactions between Hb (modified or not) and EC. We also propose a wide range of techniques and methods to assess oxidative stress and inflammation responses of EC after exposure to Hb. This editorial can serve as a guide to evaluate in vitro toxicity of new Hb molecules.