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Hydrogen peroxide is an unavoidable by-product of cell metabolism, but when it is not properly managed by the body it can lead to several pathologies (e.g., premature aging, cardiovascular and neurodegenerative diseases, cancer). Several methods have been proposed for the measurement of intracellular H2O2 but none of them has proven to be selective. We developed a rapid all-in-one chemiluminescent bioassay for the quantification of H2O2 in living cells with a low limit of detection (0.15 μM). The method relies on an adamantylidene-1,2-dioxetane lipophilic probe containing an arylboronate moiety; upon reaction with H2O2 the arylboronate moiety is converted to the correspondent phenol and the molecule decomposes leading to an excited-state fragment that emits light. The probe has been successfully employed for quantifying intracellular H2O2 in living human endothelial, colon and keratinocyte cells exposed to different pro-oxidant stimuli (i.e., menadione, phorbol myristate acetate and lipopolysaccharide). Imaging experiments clearly localize the chemiluminescence emission inside the cells. Treatment of cells with antioxidant molecules leads to a dose-dependent decrease of intracellular H2O2 levels. As a proof of concept, the bioassay has been used to measure the antioxidant activity of extracts from Brassica juncea wastes, which contain glucosinolates, isothiocyanates and other antioxidant molecules.