Tumor oxygenation is one of the key factors influencing disease prognosis and the effectiveness of treatment. Assessment of tumor oxygenation levels facilitates the selection of optimum conditions for radiation therapy, and plays an important role in creating alternative regimes of irradiation. Treating tumors with agents capable of increasing tumor oxygenation in order to increase radiosensitivity is a promising avenue of enquiry. Diffuse optical spectroscopy (DOS) allows a noninvasive determination of tissue oxygen levels based on information about the local changes in optical parameters, and the visualization of metabolic processes in the region of interest. DOS allows reconstruction of the two-dimensional distribution of main tissue chromophores that characterize the processes of oxygen supply (oxygenated hemoglobin) and oxygen consumption (deoxygenated hemoglobin), as well as the blood oxygen saturation levels, which indirectly reflect the tissue oxygenation levels. In the present study, a hemorheologic drug, pentoxifylline, which can improve microcirculation in regions with circulatory disturbances, was used for modifying tumor tissue oxygenation. Pliss’s lymph sarcoma (PLS), which is characterized by rapid growth and early occurrence of necrotic areas, was chosen as a tumor model. Tumor oxygenation was monitored by DOS with parallel plane geometry. Pentoxifylline could improve tumor oxygenation by increasing the concentration of oxyhemoglobin. The increased blood oxygen saturation persisted from 30 to 120 min after drug administration. Normal healthy tissue (muscle) and tumor tissue responded differently to the drug. DOS can be used for testing new agents that influence tissue oxygen status and blood-filling rate.