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There is a strong need for techniques that can quantify the important reactive oxygen species hydrogen peroxide (H2O2) in complex media and in vivo. We combined chemiluminescence-based H2O2 measurements on a commercially available flow injection analysis (FIA) system with sampling of the analyte using microdialysis probes (MDPs), typically used for measurements in tissue. This allows minimally invasive, quantitative measurements of extracellular H2O2 concentration and dynamics utilizing the chemiluminescent reaction of H2O2 with acridinium ester. By coupling MDPs to the FIA system, measurements are no longer limited to filtered, liquid samples with low viscosity, as sampling via a MDP is based on a dynamic exchange through a permeable membrane with a specific cut-off. This allows continuous monitoring of dynamic changes in H2O2 concentrations, alleviates potential pH effects on the measurements, and allows for flexible application in different media and systems. We give a detailed description of the novel experimental setup and its measuring characteristics along with examples of application in different media and organisms to highlight its broad applicability, but also to discuss current limitations and challenges. The combined FIA-MDP approach for H2O2 quantification was used in different biological systems ranging from marine biology, using the model organism Exaiptasia pallida (light stress induced H2O2 release up to ˜ 2.7 μM), over biomedical applications quantifying enzyme dynamics (glucose oxidase in a glucose solution producing up to ˜ 60 μM H2O2 and the subsequent addition of catalase to monitor the H2O2 degradation process) and the ability of bacteria to modify their direct environment by regulating H2O2 concentrations in their surrounding media. This was shown by the bacteria Pseudomonas aeruginosa degrading ˜ 18 μM background H2O2 in LB-broth. We also discuss advantages and current limitations of the FIA-MDP system, including a discussion of potential cross-sensitivity and interfering chemical species.Successful combination of a FIA system with microdialysis probes for H2O2 detection.Near real time, chemiluminescence detection of H2O2 dynamics in different media.Extracellular H2O2 dynamics in liquid and semisolid, untreated samples.System tunable to different calibration ranges (0–1000 nM; 0–10 μM; 1–100 μM).Cross-sensitivity study of potentially interfering species and the chemiluminescent reaction.