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The detection of radical oxygen species (ROS) is central to the understanding of their role in signal transduction. ROS detection is based on the radical-dependent reduction of a compound with a measurable change in a chemical property. However, in vitro several of the compounds are reduced by several oxidants, which results in a lack of specificity when used to detect intracellular ROS. In contrast, by using methods that detect extracellular ROS, specificity can be corroborated by adding the appropriate competitor; for example, superoxide dismutase competes for superoxide, and catalase competes for H2O2. In addition, because of the efficient activity of superoxide dismutases on superoxide and H2O2 membrane permeability, determination of extracellular H2O2 can detect all potential cellular sources of these ROS. In this protocol, extracellular H2O2 is measured as the limiting factor of peroxidase-mediated oxidation of homovanillic acid into a fluorescent dimer. The specificity of this reaction for H2O2 is demonstrated by the addition of catalase as an H2O2 scavenger. Because the assay detects small changes in fluorescence, it is highly sensitive. The high sensitivity and the specificity of this assay make it well suited to measure ROS in nonphagocytic cells where the ROS levels are in the low micromolar range. To further increase sensitivity, H2O2 measurements are performed over time to ensure the detection of maximum response and to minimize the variability in response arising from cellular heterogeneity, an attribute of primary cultures.