lysosomal cysteine proteinase cathepsin b (ctb) is a member of the cysteine protease family known to participate in intracellular degradation processes and protein turnover in the lysosomes of healthy cells. cathepsin b plays a crucial role in tumor invasion and progression by controlling extracellular degradation and participating in a proteolytic cascade activation (gondi and rao 2013). its role in tumor invasion and progression makes ctb a promising biomarker and target for antibody-directed prodrug therapy (dheer, nicolas et al. 2019). the development of novel ctb-specific molecular probes opens new possibilities for image-based diagnostic methods for different types of cancers (podgorski and sloane 2003, tan, peng et al. 2013). since aberrant expression of this protein has been an indicator of cancer development, detecting ctb expression and activity might be beneficial for the early detection of cancer or revealing aggressive lesions (gondi and rao 2013). developing probes capable of binding with ctb is challenging due to binding site homology to other members of the cysteine cathepsin family (turk, stoka et al. 2012). in this study, to identify unique residues in human ctb compare to other members of the cathepsin family, amino acid sequences of these proteins were exposed to. multiple sequence alignments. cathepsin b in humans has three active site residues critical for catalysis: cys108; his278, and asp298 (ruan, hao et al. 2015) were confirmed with multiple sequence alignment as fully conserved residues. the initial step in the development of a detection assay for ctb is finding appropriate fluorescent small molecules for enzyme binding. in this study, two ligand candidates cid8795 and cid535684 were identified and successfully conjugated to the dye atto680 and were tested for binding affinity and specificity to ctb. for cid535684atto680, no binding interaction was observed in the fluorescence polarization (fp) assay. cid8795atto680 demonstrated increases in fluorescence polarization assays in the presence of ctb with the half-maximal effective concentration (ec50) at 3.27 ± 1.27 nm. a third probe, benzyloxycarbonyl (cbz)-lys-lys-p-aminobenzyl alcohol (paba)-2’, 7’-dichloro-6’-methoxy-fluorescein (dcmf), was designed based on a known substrate scaffold for ctb. this novel substrate-based fluorescent probe was shown to be hydrolyzed by ctb having a specificity constant kcat/km = 41.9 ± 0.07 mm-1 × s-1 . finally, we investigated single nucleotide polymorphisms (snps) within the coding region of the ctsb gene within the general population (random data from 2,504 samples) included in the 1,000 genomes project. the mapping of snps onto the 3d structure of cathepsin b indicates that the active site of ctb is fully conserved among humans – as no snps were identified within the binding pocket of ctb. according to these results, probes that bind to the enzyme’s active site should be generally useful for detecting ctb in all populations studied in the 1,000 genomes project.