Cathepsin B (CTSB, EC 22.214.171.124) is a lysosomal cysteine proteinase that is suggested to participate in intracellular degradation and turnover of proteins. It has also been implicated in tumor invasion and metastasis. The Cathepsin B Inhibitor Screening Kit utilizes the ability of Cathepsin B 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 B-specific inhibitor, the cleavage of the 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 the potential inhibitors of Cathepsin B.