Hypoxic lesions often respond poorly to cancer therapies. Particularly, photodynamic therapy (PDT) consumes oxygen in treated tissues, which in turn lowers its efficacy. Tools for online monitoring of intracellular pO2 are desirable. The pO2 changes were tracked during photodynamic therapy (PDT) with δ-aminolevulinic acid (ALA) in mouse skin, xenograft tumors, and human skin. ALA was applied either topically as Ameluz cream or systemically by injection. Mitochondrial pO2 was quantified by time-gated lifetime-based imaging of delayed fluorescence (DF) of protoporphyrin IX (PpIX). pO2-weighted images were obtained with capture-times of several seconds, radiant exposures near 10 mJ/cm2, spatial resolution of 0.3 mm, and a broad dynamic range 1-50 mmHg, corresponding to DF lifetimes ≈20-2000 μs. The dose-rate effect on oxygen consumption was investigated in mouse skin. A fluence rate of 1.2 mW/cm2 did not cause any appreciable oxygen depletion, whereas 6 mW/cm2 and 12 mW/cm2 caused severe oxygen depletion after radiant exposures of only 0.4-0.8 J/cm2 and <0.2 J/cm2, respectively. Reoxygenation after PDT was studied too. With a 5 J/cm2 radiant exposure, the recovery times were 10-60 min, whereas with 2 J/cm2 they were only 1-6 min. pO2 distribution was spatially non-uniform at (sub)-millimeter scale, which underlines the necessity of tracking pO2 changes by imaging rather than point-detection. Time-gated imaging of PpIX DF seems to be a unique tool for direct online monitoring of pO2 changes during PDT with a promising potential for research purposes as well as for comparatively easy clinical translation to improve efficacy in PDT treatment.