question: Reactive molecular oxygen (O2) plays important roles in bioenergetics and metabolism and is implicated in biochemical pathways underlying angiogenesis, fertilization, wound healing and regeneration. Here we describe how to use the scanning micro-optrode technique (SMOT) to measure extracellular fluxes of dissolved O2. The self-referencing O2-specific micro-optrode (also termed micro-optode and optical fiber microsensor) is a tapered optical fiber with an O2-sensitive fluorophore coated onto the tip. The O2 concentration is quantified by fluorescence quenching of the fluorophore emission upon excitation with blue-green light. The micro-optrode presents high spatial and temporal resolutions with improved signal-to-noise ratio (in the picomole range). In this protocol, we provide step-by-step instructions for micro-optrode calibration, validation, example applications and data analysis. We describe how to use the technique for cells (Xenopus oocyte), tissues (Xenopus epithelium and rat cornea), organs (Xenopus gills and mouse skin) and appendages (Xenopus tail), and provide recommendations on how to adapt the approach to different model systems. The basic, user-friendly system presented here can be readily installed to reliably and accurately measure physiological O2 fluxes in a wide spectrum of biological models and physiological responses. The full protocol can be performed in ~4 h.