AbstractThe last two decades have witnessed a major revolution in the field of tumor immunology including clinical progress using various immunotherapy strategies. These advances have highlighted the potential for approaches that harness the power of the immune system to fight against cancer. While cancer immunotherapies have shown significant clinical successes, patient responses vary widely due to the complex and heterogeneous nature of tumors and immune responses, calling for reliable biomarkers and therapeutic strategies to maximize the benefits of immunotherapy. Especially, stratifying responding individuals from non‐responders during the early stages of treatment could help avoid long‐term damage and tailor personalized treatments. In efforts to develop non‐invasive means for accurately evaluating and predicting tumor response to immunotherapy, multiple affinity‐based agents targeting immune cell markers and checkpoint molecules have been developed and advanced to clinical trials. In addition, researchers have recently turned their attention to substrate and activity‐based imaging probes that can provide real‐time, functional assessment of immune response to treatment. Here, we highlight some of those recently designed probes that image functional proteases as biomarkers of cancer immunotherapy with a focus on their chemical design and detection modalities and discuss challenges and opportunities for the development of imaging tools utilized in cancer immunotherapy.