A method for correctly assessing hydroxyl radical scavenging activity of antioxidative chemicals and/or biological compounds/materials was proposed. This method can simultaneously assess two factors, i.e. hydroxyl radical-scavenging and 5,5-dimethyl-2-hydroxy-1-pyrrolidine- N -oxide (hydroxyl radical adduct of 5,5-dimethyl-1-pyrroline- N -oxide)-reducing ability, as antioxidative properties. In this paper, some biologically common hydrophilic molecules, cell culture media, and rat plasma were tested. X-ray-induced hydroxyl radical can be detected using the electron paramagnetic resonance spin trapping technique. Using X-ray irradiation of the reaction mixture as the hydroxyl radical source, the true hydroxyl radical-scavenging ability of the subjected antioxidant can be assessed. In addition, the method simultaneously measures the reduction of 5,5-dimethyl-2-hydroxy-1-pyrrolidine- N -oxide, to estimate the reducing ability of the antioxidant. Biological materials, such as sugars and proteins, could abolish hydroxyl radical at the biological concentration. Ascorbic acid showed reducing ability at the biological concentration. The simultaneous assessment of hydroxyl radical-scavenging and reducing ability of antioxidants can be an informative index for antioxidants.