Poor oxygenation of solid tumours has been linked with resistance to chemo- and radio-therapy and poor patient outcomes. Measuring the functional status of the tumour vasculature, including blood flow fluctuations and changes in oxygenation is important in cancer staging and therapy monitoring. A robust method is needed for clinical non-invasive measurement of the oxygen supply and demand in tumours. Current clinically approved imaging modalities suffer high cost, long procedure times and limited spatio-temporal resolution. Optoacoustic tomography (OT) is an emerging clinical imaging modality that can provide static images of endogenous haemoglobin concentration and oxygenation. In this work, an integrated framework for quantitative analysis of functional imaging using OT is developed and applied in vivo with preclinical cancer models. Oxygen Enhanced (OE)-OT is established here to provide insight into tumour vascular function and oxygen availability in the tissue. Tracking oxygenation dynamics using OE-OT reveals significant differences between two prostate cancer models in nude mice with markedly different vascular function (PC3 & LNCaP), which appear identical in static OT. OE-OT metrics are shown to be highly repeatable and correlate directly on a per-tumour basis to tumour vascular maturity, hypoxia and necrosis, assessed ex vivo. Dynamic Contrast Enhanced (DCE) OT demonstrates the relationship between OE-OT response and tumour perfusion in vivo. Finally, the possibility of using OT data acquired at longer wavelengths to report on tumour water and lipid content is investigated, with a view to future providing intrinsically co-registered imaging of tumour oxygenation and cellular necrosis. These findings indicate that OE-OT holds potential for application in prostate cancer patients, to improve delineation of aggressive and indolent disease, while combined with DCE-OT, it may offer significant advantage for localised imaging of tumour response to vascular targeted therapies. Further work is needed to establish whether OT can provide a new method to detect tumour necrosis in vivo.