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Reactive Oxygen Species (ROS) in the form of H 2 O 2 can act both as physiological signaling molecules as well as damaging agents, depending on their concentration and localization. The downstream biological effects of H 2 O 2 were often studied making use of exogenously added H 2 O 2 , generally as a bolus and at supraphysiological levels. But this does not mimic the continuous, low levels of intracellular H 2 O 2 production by for instance mitochondrial respiration. The enzyme d-Amino Acid Oxidase (DAAO) catalyzes H 2 O 2 formation using d-amino acids, which are absent from culture media, as a substrate. Ectopic expression of DAAO has recently been used in several studies to produce inducible and titratable intracellular H 2 O 2 . However, a method to directly quantify the amount of H 2 O 2 produced by DAAO has been lacking, making it difficult to assess whether observed phenotypes are the result of physiological or artificially high levels of H 2 O 2 . Here we describe a simple assay to directly quantify DAAO activity by measuring the oxygen consumed during H 2 O 2 production. The oxygen consumption rate (OCR) of DAAO can directly be compared to the basal mitochondrial respiration in the same assay, to estimate whether the ensuing level of H 2 O 2 production is within the range of physiological mitochondrial ROS production. In the tested monoclonal RPE1-hTERT cells, addition of 5 mM d-Ala to the culture media amounts to a DAAO-dependent OCR that surpasses ∼5% of the OCR that stems from basal mitochondrial respiration and hence produces supra-physiological levels of H 2 O 2 . We show that the assay can also be used to select clones that express differentially localized DAAO with the same absolute level of H 2 O 2 production to be able to discriminate the effects of H 2 O 2 production at different subcellular locations from differences in total oxidative burden. This method therefore greatly improves the interpretation and applicability of DAAO-based models, thereby moving the redox biology field forward., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)