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Reactive oxygen species are ubiquitous in nature, and function as signalling molecules in biological systems; they may also contribute to oxidative stress in several pathobiological disease states. In this report, we describe a simple, reliable, sensitive, and specific assay for the detection and quantitation of hydrogen peroxide (H2O2) release by living cells, organoids, or tissues. Furthermore, the low cost of reagents required for this assay makes it inexpensive relative to commercial kits. The high sensitivity and specificity are based on the ability of H2O2 to react with heme peroxidases and convert para-substituted phenolic compounds to fluorescent dimers.Graphical abstract: