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Myoglobin is an essential oxygen-binding hemoprotein in skeletal and cardiac muscles that buffers intracellular oxygen () concentration in response to hypoxia or elevated muscle activities. We present a method that uses photoacoustic computed tomography to measure the distribution of myoglobin in tissue and the oxygen saturation of myoglobin (). From photoacoustic measurements of mice in different oxygenation states, we performed calibration-free quantification of the change in the backbone muscle in vivo.