Natural killer (NK) cells are critical in diverse defense mechanisms, including elimination of viral infected cells and destruction of tumor cells. NK cells are characterized by the ability to initiate apoptosis in target cells when their cell surface major histocompatibility complex class I (MHC I) repertoire is missing. On the other hand, NK cells are not activated when MHC I or non-classical MHC molecules are found on the respective cells. It was demonstrated that cathepsin G (CatG) binds to the cell surface of NK cells; however, the distribution of this protease on the cell surface of NK cell subsets has not been identified. Here, we show that CatG cell surface level differs between NK cell subsets. CatG was determined on the protein- and activity level (activity-based probe MARS116) by using flow cytometry. Thus, MARS116 is a novel reporter of cell surface CatG activity and can be used to differentiate between distinct NK cell subsets.