Radiotherapy is commonly used for cancer therapy, although its efficacy is reduced in hypoxic regions of tumours. Photoacoustic imaging (PAI) is an emergent, non-invasive imaging technique that allows the measurement of blood oxygen saturation (sO 2 ) which inversely correlates with hypoxia in tissue. The potential use of PAI as a prognostic tool for radiotherapy outcome was investigated in a head and neck cancer model in vivo. PAI was performed before delivering a single fraction (10, 20 or 30 Gy) treatment. The results show that tumours with pre-treatment higher blood sO 2 responded better than those with lower levels in the 10 and 20 Gy groups. For the 30 Gy group, treatment response was independent of blood sO 2 . The haemoglobin content of the tumours was not correlated with their response to any of the radiation doses studied. Changes in sO 2 , monitored at 24 h and 96 h following 10 and 20 Gy doses, showed that tumours that were subsequently unresponsive to treatment had an increase in blood sO 2 at both time points compared to those which subsequently regressed after radiotherapy. The results suggest that sO 2 values measured by photoacoustic imaging can be used before, and shortly after, irradiation to predict subsequent treatment response.