Cathepsin K (CTSK, EC 220.127.116.11), a lysosomal cysteine protease, is involved in osteoclastic bone remodeling and resorption. In addition, it also degrades collagen, gelatin and elastin. The Cathepsin K Inhibitor Screening Kit utilizes the ability of active Cathepsin K to cleave the synthetic AFC based peptide substrate to release AFC, which can be easily quantified using a fluorometer or fluorescence microplate reader. In the presence of a Cathepsin K-specific inhibitor, the cleavage of this substrate is reduced/abolished resulting in decrease or total loss of the AFC fluorescence. This simple and high-throughput adaptable assay kit can be used to screen/study/characterize potential inhibitors of Cathepsin K.