Acidic extracellular pH (pHe) of gliomas promotes tumor growth and builds resistance to therapy. Thus, to monitor therapeutic response, we used Biosensor Imaging of Redundant Deviation in Shifts (BIRDS), to generate pHe maps of rat brains bearing U251 tumors. Upon TmDOTP5- infusion, MRI identified tumor boundary and BIRDS imaged the intratumoral and peritumoral pHe gradient (DpHe). Two weeks post implantation of U251 glioma cells, animals were either treated with temozolomide (40 mg/kg) or were left untreated. The results of both terminal and longitudinal studies suggest that temozolomide therapy hinders tumor growth and normalizes intratumoral pHe.