The aim of this study was to validate orthogonal polarization spectral (OPS) imaging against intravital fluorescence microscopy (IFM) for microvascular measurements in normal skin and during wound healing. Experiments were performed on the ears of hairless mice (N = 8). The diameter of arterioles and venules, red blood cell velocity in venules, and the functional capillary density were assessed under normal conditions using OPS imaging and IFM. After creation of a circular wound, these observations were repeated at the identical microvascular regions on days 4, 7, 10, and 15. Images were videotaped, and CapImage was used for off-line computer-assisted analysis. Using OPS imaging, the microcirculation of wounded skin in hairless mice could be observed successfully. The regression analyses against standard IFM revealed a significant (p < 0.001) correlation for measurements of all microcirculatory parameters investigated (venular diameter:r2 = 0.98, N = 345; red blood cell velocity:r2 = 0.51, N = 326; functional capillary density:r2 = 0.44, N = 156). However, for diameter as well as for functional capillary density measurements, OPS imaging yielded lower absolute values compared with IFM. The authors were able to validate OPS imaging against IFM for the measurement of microvascular parameters in an animal model of skin wound healing. Such a device should now help to study the role of microcirculation in physiology and pathophysiology during wound healing in patients. First clinical investigations are promising.