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Leaf metabolism produces hydrogen peroxide (H2O2) at high rates, high level H2O2 accumulation can cause oxidative stress. This protocol describes a method for determining H2O2 concentration in tobacco leaves. In this method all extractions were performed with HClO4, neutralized, and pretreated with ascorbate oxidase to eliminate ascorbate interferences. H2O2 content was determined using a colorimetric assay spiked with an internal control. Interfering peroxides were determined in parallel using a negative control treated with catalase and subsequently subtracted.