Checkpoint inhibitors potentially could be used to enhance cell killing by DNA-targeted therapeutic modalities such as radiotherapy. UCN-01 (7-hydroxystaurosporine) inhibits S and G₂ checkpoint arrest in the cells of various malignant cell lines and has been investigated in combination with chemotherapy. However, little is known about its potential use in combination with radiotherapy. We report the effect of 20 Gy radiation given in conjunction with UCN-01 on the pO₂ and growth of subcutaneous RIF-1 tumors. Multisite EPR oximetry was used for repeated, non-invasive tumor pO₂ measurements. The effect of UCN-01 and/or 20 Gy on tumor pO₂ and tumor volume was investigated to determine therapeutic outcomes. Untreated RIF-1 tumors were hypoxic with a tissue pO₂ of 5–7 mmHg. Treatment with 20 Gy or UCN-01 significantly reduced tumor growth, and a modest increase in tumor pO₂ was observed in tumors treated with 20 Gy. However, irradiation with 20 Gy 12 h after UCN-01 treatment resulted in a significant inhibition of tumor growth and a significant increase in tumor pO₂ to 16–28 mmHg from day 1 onward compared to the control, UCN-01 or 20-Gy groups. Treatment with UCN-01 12 h after 20 Gy also led to a similar growth inhibition of the tumors and a similar increase in tumor pO₂. The changes in tumor pO₂ observed after the treatment correlated inversely with the tumor volume in the groups receiving UCN-01 with 20 Gy. This multimodal approach could be used to enhance the outcome of radiotherapy. Furthermore, tumor pO₂ could be a potential marker of therapeutic response.