Quantification of blood oxygen saturation (SO2) in vivo is essential for understanding the pathogenesis of diseases in which hypoxia is thought to play a role, including inflammatory disorders such as multiple sclerosis (MS) and rheumatoid arthritis (RA). We describe a low-cost multispectral microscope and oximetry technique for calibration-free absolute oximetry of surgically exposed blood vessels in vivo. We imaged the vasculature of the dorsal spinal cord in healthy rats, and varied inspired oxygen (FiO2) in order to evaluate the sensitivity of the imaging system to changes in SO2. The venous SO2 was calculated as 67.8 ± 10.4% (average ± standard deviation), increasing to 83.1 ± 11.6% under hyperoxic conditions (100% FiO2) and returning to 67.4 ± 10.9% for a second normoxic period; the venous SO2 was 50.9 ± 15.5% and 29.2 ± 24.6% during subsequent hypoxic states (18% and 15% FiO2 respectively). We discuss the design and performance of our multispectral imaging system, and the future scope for extending this oximetry technique to quantification of hypoxia in inflamed tissue.