Acute lymphoblastic leukemia (ALL) interacts with bone marrow cells, creating hypoxic niches that stabilize HIF-1α and promote chemotherapeutic resistance. Spectrosocopic photoacoustic (PA) imaging is a label-free, noninvasive technique that probes the <i>in vivo</i> oxygenation status of hemoglobin, resulting in a measurement of oxygen saturation (SO<sub>2</sub>) and providing a surrogate measure of tissue hypoxia. This work investigates multispectral PA imaging to assess the SO<sub>2</sub> in the femoral bone marrow in mice. Preliminary work was performed to assess the capability of imaging through bone, followed by an oxygen challenge to determine the magnitude of systemic SO<sub>2</sub> changes measurable in wild type mice. Furthermore, a pilot study to compare SO<sub>2</sub> measured in a murine model of ALL versus in healthy controls was performed to investigate a correlation between SO<sub>2</sub> changes in the femoral bone marrow and disease progression. Study results show that femoral SO<sub>2</sub> can be measured with a variation less than 10% in wild type mice over multiple time-points. In the oxygen challenge, a 10% difference in systemic SO<sub>2</sub> was observed between 100% and 21% O<sub>2</sub> inhalation conditions. Additionally, leukemic mice demonstrate significantly more variation in femoral SO<sub>2</sub> over the length of the femur than control mice at day 14 post-inoculation, indicating that femoral SO<sub>2</sub> is affected by leukemic disease progression. This work demonstrates the feasibility of observing changes in leukemic disease progression through the measurement of SO<sub>2</sub> with spectroscopic PA imaging, which could help develop a more complete understanding of the interplay of the local microenvironment with leukemogenesis.