Immature neovessels in tumors lead to the formation of heterogeneous hypoxic areas within tumors. Such hypoxic conditions not only reduce the effectiveness of radiation treatment but are also related to tumor invasion and metastasis. To better understand tumor-related mechanisms, it is important to quantitate the temporal and spatial changes of tumor hypoxia. However, no useful technique capable of measuring oxygen concentration in vivo has been proposed to date. We aimed to develop a microscope system to measure oxygen metabolism in tumor tissues using a laser-assisted oxygen quenching method. A scanning laser with galvano mirrors was capable of imaging tissue hypoxia and allowed configuration of time and spatial resolution by altering the laser spot size, scan rate, or scan distance. We examined the feasibility of the system by in vitrooxygen measurement, and applied this method to in vivo imaging of tumor oxygenation during oxygen inhalation in tumor-implanted mice. Oxygen tension inside tumors increased soon after oxygen inhalation, but decreased gradually after 20 min in spite of continuous oxygen inhalation, indicating that this model replicates the clinical experience that long-term tumor oxygenation cannot be achieved by oxygen inhalation. Quantitative analysis of tumor oxygenation may help reveal the mechanisms of oxygen metabolism in tumor tissues, leading to the development of more effective radiation therapy.