AbstractMeasuring oxygen concentration in three‐dimensional cultures, without interfering with cellular activities, is a fundamental request of tissue engineering research. Among the other techniques, it has been demonstrated that phosphorescence quenching microscopy (PQM) represents a valid tool for the detection of oxygen concentration in 3D environments. Indeed, it is not invasive, with high spatial and temporal resolution, and, once calibrated, it is not affected by the presence of extracellular matrix components and other environmental factors. In this work, a description of the PQM experimental set up for oxygen measurements in solutions and 3D polymer‐based cellular constructs is provided. Moreover, the advantage and the limits in the use of this technique are critically discussed to provide a technical note for future applications. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010