Background: Knowledge of tissue oxygenation status is fundamental in the prevention of postoperative flap failure. Recently, the authors introduced a novel oxygen-sensing paint-on bandage that incorporated an oxygen-sensing porphyrin with a commercially available liquid bandage matrix. In this study, the authors extend validation of their oxygen-sensing bandage by comparing it to the use of near-infrared tissue oximetry in addition to Clark electrode measurements. Methods: The oxygen-sensing paint-on bandage was applied to the left hind limb in a rodent model. Simultaneously, a near-infrared imaging device and Clark electrode were attached to the right and left hind limbs, respectively. Tissue oxygenation was measured under normal, ischemic (aortic ligation), and reperfused conditions. Results: On average, the oxygen-sensing paint-on bandage measured a decrease in transdermal oxygenation from 85.2 mmHg to 64.1 mmHg upon aortic ligation. The oxygen-sensing dye restored at 81.2 mmHg after unclamping. Responses in both control groups demonstrated a similar trend. Physiologic changes from normal to ischemic and reperfused conditions were statistically significantly different in all three techniques (p < 0.001). Conclusions: The authors' newly developed oxygen-sensing paint-on bandage exhibits a comparable trend in oxygenation recordings in a rat model similar to conventional oxygenation assessment techniques. This technique could potentially prove to be a valuable tool in the routine clinical management of flaps following free tissue transfer. Incorporating oxygen-sensing capabilities into a simple wound dressing material has the added benefit of providing both wound protection and constant wound oxygenation assessment.