Perfusion-limited hypoxia, referring to spatial and temporal fluctuations in oxygen levels, has emerged as a key driver of malignant disease progression in cancer, promoting higher metastatic potential, increased therapy resistance and poorer patient outcome1,2. However, the origin and impact of these hypoxic fluctuations on tumour progression have not yet been fully understood. Harnessing the photoacoustic effect, photoacoustic imaging (PAI) holds significant potential to elucidate these dynamics, but to fulfil this potential, a need for a thorough technical and biological validation arises. This thesis presents technical validation of PAI systems, which builds confidence in the subsequent biological validation performed using relevant biomarkers in the studies of perfusion-limited hypoxia. First, the current state-of-the art phantom materials in biophotonics are surveyed and general design considerations for preparation of tissue-mimicking phantoms are discussed to guide the development of a stable phantom material in PAI. Acoustic and optical material characterisation systems are then established and validated to enable thorough characterisation of phantom materials. Building up on this groundwork, a phantom material is developed for use in PAI, which exhibits stable acoustic, optical and mechanical properties with tuneable tissue-mimicking characteristics. Using custom phantom setups, a thorough technical characterisation study of a commercial mesoscopic PAI system is then conducted, outlining strengths and limitations of the system in characterising vessel-related biomarkers in tissues. Following these technical validation studies, the thesis embarks on applying photoacoustic mesoscopy and macroscopy in studies of perfusion-limited hypoxia in two distinct murine xenograft models, and validating this work using histopathological and transcriptomic analyses. These studies indicate that tumour vasculature undergoes rapid fluctuations in perfusion that are impacted by the underlying maturity of the vascular network, leading to variations in tumour oxygenation. Building up on an extensive validation framework, this thesis highlights the promise of PAI to advance our understanding on perfusion- and oxygenation dynamics in tumour tissues, thereby assisting the development of targeted treatment regimes in future.