The serine synthesis pathway (SSP), which provides precursors for redox homeostasis and nucleotide synthesis, has emerged as a critical metabolic pathway in cancer. However, the assessment of therapeutic approaches targeting the SSP has been challenging due to a lack of distinct biomarkers. We have identified that the SSP inhibition increases reactive oxygen species (ROS) levels and, intriguingly, glycolytic rate in leukemia cells. Using hyperpolarized dehydroascorbate and pyruvate magnetic resonance, we assessed therapeutic responses earlier than any significant changes in cell viability. This approach has broad implications as an effective methodology for monitoring therapeutic responses with SSP inhibition in multiple cancers.