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Natural killer (NK) cells are innate immune cells that can directly kill certain types of cancer cells. Adoptive transfer of NK cells represents a novel and promising immunotherapy for malignant tumours; however, there is a lack of methods to validate anti‐tumour activity of NK cells reaching target sites in vivo. Herein we report a new chemiluminescent probe to image in situ the granzyme B‐mediated killing activity of NK cells against cancer cells. We have optimised a granzyme B‐specific construct using an activatable phenoxydioxetane reporter so that enzymatic cleavage of the probe results in bright chemiluminescence in the visible range. The probe shows high selectivity for active granzyme B over other proteases and higher signal‐to‐noise ratios than commercial fluorophores. Finally, we demonstrate that the probe can detect activity of NK cells in preclinical mouse models, being the first chemiluminescent probe for in vivo imaging of NK cell activity in live tumours.