Recent years attracted high interest in research toward natural-killer cells (NK) and cytotoxic lymphocyte T cells (CLTs) due to their involvement in bacteria killing and anticancer therapies associated with tumor cell death. In these events granzymes (Grs), which belong to serine proteases family are considered to be a key players. Grs are released by NK and CLTs in order to eliminate cancer cells and bacteria or viruses infected cells. There are five Grs in humans, however despite intense research, precise role of all Grs has not been explained yet in detail, mainly due to the lack of specific tools for their investigation. In this interdisciplinary project we aim to engineer new “smart activity-based probes”, which produce strong fluorescence signal when specifically cleaved by targeted protease (fluorogenic “turn on” probes) and apply them to answer important questions related to functions and localization of Grs using different imaging techniques.