Myoglobin is an essential oxygen-binding hemoprotein in skeletal and cardiac muscles that buffers intracellular oxygen (O2) 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 (sO2-Mb ). From photoacoustic measurements of mice in different oxygenation states, we performed calibration-free quantification of the sO2-Mb change in the backbone muscle in vivo.