In vivo quantification of extracellular pH (pHe) in tumour may provide a useful biomarker for tumour cell metabolism. In this study, we assess the viability of continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy with a pH-sensitive nitroxide to measure extracellular tumour pH in the mouse model. 750 MHz CW-EPR spectroscopy of C3H HeJ mice hind leg squamous cell tumour was performed after intra-venous tail vein injection of pH-sensitive nitroxide (R-SG) during stages of normal tumour growth, and in response to a single 10 Gy dose of X-ray irradiation. An inverse relationship was observed between tumour volume and pHe value, whereby during normal tumour growth a constant reduction in pHse was observed. This relationship was disrupted by X-ray irradiation, and from 2–3 days post exposure, a transitory increase pHe was observed. In this study we demonstrated the viability of CW-EPR spectroscopy using R-SG nitroxide to obtain high sensitivity pH measurements in mouse tumour model with accuracy < 0.1 pH units. In addition, measured changes in pHe in response to X-ray irradiation suggest this may offer a useful method for assessing physiological change in response to existing and novel cancer therapies.