This study investigates the correlation of tumor blood oxygenation and tumor pO2 with respect to carbogen inhalation. After having refined and validated the algorithms for calculating hemoglobin concentrations, we used near-infrared spectroscopy (NIRS) to measure changes of oxygenated hemoglobin concentration (Δ[HbO2]) and used an oxygen needle electrode and 19F MRI for pO2 measurements in tumors. The measurements were taken from Dunning prostate R3327 tumors implanted in rats, while the anesthetized rats breathed air or carbogen. The NIRS results from tumor measurements showed significant changes in tumor vascular oxygenation in response to carbogen inhalation, while the pO2 electrode results showed an apparent heterogeneity for tumor pO2 response to carbogen inhalation, which was also confirmed by 19F MR pO2 mapping. Furthermore, we developed algorithms to estimate hemoglobin oxygen saturation, sO2, during gas intervention based on the measured values of Δ[HbO2] and pO2. The algorithms have been validated through a tissue-simulating phantom and used to estimate the values of sO2 in the animal tumor measurement based on the NIRS and global mean pO2 values. This study demonstrates that the NIRS technology can provide an efficient, real-time, noninvasive approach to monitoring tumor physiology and is complementary to other techniques, while it also demonstrates the need for an NIR imaging technique to study spatial heterogeneity of tumor vasculature under therapeutic interventions.