•A clickable and tagless activity-based probe (ABP) of human cathepsin B, KDA-1, is developed with a minimalistic design approach for enhanced selectivity.•The probe KDA-1 is highly selective, and targets the active site Cys29 residue for labeling in an activity-dependent manner.•A cell permeable analogue, KDA-1-OMe has also been developed and characterized.•Cathepsin B probe, KDA-1, can successfully label the active enzyme from proteomes derived from human MDA-MB-231 breast cancer cells and HEK293 cells. Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.