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 in vivo oxygenation status of hemoglobin, resulting in a measurement of oxygen saturation (SO2) and providing a surrogate measure of tissue hypoxia. This work investigates multispectral PA imaging to assess the SO2 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 SO2 changes measurable in wild type mice. Furthermore, a pilot study to compare SO2 measured in a murine model of ALL versus in healthy controls was performed to investigate a correlation between SO2 changes in the femoral bone marrow and disease progression. Study results show that femoral SO2 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 SO2 was observed between 100% and 21% O2 inhalation conditions. Additionally, leukemic mice demonstrate significantly more variation in femoral SO2 over the length of the femur than control mice at day 14 post-inoculation, indicating that femoral SO2 is affected by leukemic disease progression. This work demonstrates the feasibility of observing changes in leukemic disease progression through the measurement of SO2 with spectroscopic PA imaging, which could help develop a more complete understanding of the interplay of the local microenvironment with leukemogenesis.