Processes of angioadaptation, like sprouting or/and degeneration of vessels result in altered distribution of oxygen. Several methods have been developed to determine oxygen tension or hemoglobin saturation (SO2) in microvessels in order to access the vital tissue status. We have developed a multispectral imaging approach, which allows the determination of oxygen saturation in microvascular networks during intravital microscopy by use of a commercially available imaging system including a tunable monochromatic light source (T.I.L.L. Photonics GmbH). Our method is based on the different absorption spectra of oxygenated and deoxygenated hemoglobin. By this technique, SO2 and hematocrit, can be obtained as 2D image of the observed area. For the validation of the system, we performed in vivo experiments (rat mesentery): hyperoxia (inspiratory O2 concentration 100%, mesentery exposed to pure oxygen), normoxia (breathing room air, mesentery covered with impermeable film), hypoxia (insp. O2 conc. 9% in N2, mesentery exposed to pure nitrogen), anoxia (superfusion of mesentery with Na-dithionite). Using data obtained by hyperoxic and anoxic conditions as references, SO2 values averaged ~95% in arterioles and ~87% in venules. Our method allows parallel in vivo estimation of SO2 and hematocrit for all vessels in a microscopic field of view as well as intravascular gradients.