Many studies have established a role for oxidative stress and mitochondrial dysfunction as an important mechanism in the pathogenesis of neuronal disorders. Metalloporphyrins are a class of catalytic antioxidants that are capable of detoxifying a wide range of reactive oxygen species. The AEOL112 series of glyoxylate metalloporphyrins were designed with increased lipid solubility for better oral bioavailability and penetration of the blood-brain barrier. The goal of this study was to develop an in vitro assay using rat brain mitochondria to reliably detect endogenously released hydrogen peroxide (H(2)O(2)) and identify glyoxylate metalloporphyrins based on rank order of potency for removal of physiologically relevant H(2)O(2). A polarographic method was established for the sensitive, accurate, and reproducible detection of low levels of H(2)O(2). The assay identified several potent glyoxylate metalloporphyrins with H(2)O(2) scavenging potencies (IC(50)) in the nanomolar range. These results provide a simplified in vitro model system to detect physiologically generated mitochondrial H(2)O(2) as a screening tool to predict the biological efficacy of potential therapeutic entities.