Even though oxygen plays a crucial role in body function and cancer biology, methods of measuring oxygen level in tissue are all limited. The current gold standard relies on an invasive electrode for only single-point reading at a time. The photoacoustic lifetime imaging (PALI) approach overcomes these major limitations by applying photoacoustic probing to oxygen-sensitive optical transient absorption. The capability of assessing oxygen distribution is demonstrated by imaging tumor hypoxia in a small animal model, and monitoring changes of tissue oxygen induced by external modulations. Proposed applications of this imaging technique includes imaging-guided photodynamic therapy (PDT) and activatable probes for molecular imaging.