Liver Fluke (Fasciola hepatica and F. gigantica) infection causes fasciolosis or Liver Rot disease, which is estimated to cause losses to the agricultural sector in the order of US$10 billion per year and is growing in significance as a neglected tropical disease that infects 17 million people worldwide. Following the accidental ingestion of metacercariae, infective stage liver fluke excyst, penetrate the duodenum of the host and migrate through the liver towards the bile ducts. During the process of invasion, liver fluke excrete/secretes multiple proteolytic enzymes that are key virulence proteins that operate at the host-parasite interface. Prominent amongst the virulence proteins released by liver fluke are cathepsin 8 proteases; these proteases not only facilitate excystment and invasion of the duodenal wall but also take part in feeding, digestion, and immune evasion. Until recently only 10 clades of cathepsin B proteases were reported, however our integrated PCR and bioinformatic-based approaches have uncovered new data on liver fluke cathepsin B proteases. Based on our findings, we propose a new clade-based classification system for cathepsin 8 proteases of the family Fasciolidae. Expression analyses identified a total of 22 cathepsin B proteases across the key life stages (metacercariae, newly excysted juvenile [NEJ] and adult) of three species, Fasciola hepatica, Fasciola gigantica and Fascioloides magna. End point PCR analyses and real-time PCR based approaches were used to interrogate the relative expression of all the cathepsin B clades identified. The results \showed that almost all the cathespin 8 clades are activated during the dormant metacercarial stage. Cathepsin B2 is highly expressed in metacercaria and NEJs but is weakly expressed in adult worms. The latter has been widely studied as a key protease in the early infection stages and has been touted as a potential control target for fluke. We also identified inconsistencies in the naming of cathepsin Bs and, based on key structrual features, we propose that F.hepatica cathepsin B1 is renamed as cathepsin B5; its homologue, FgCB5, was found to be expressed in F. gigantica. The putative cathepsins B5 and B8 (Robinson et. al., 2009) were found to be part of cathepsins 87 and 86, respectively and have been redesignated accordingly. A homologue of cathepsin 87 (FmCB7) was also discovered in F. magna and it was more like FgCB7 than FhCB7. Some cathepsin 8 proteases from fluke have been proposed to be inactive, e.g. F. hepatica cathepsin 84 (FhCB4) because it possess possesses an active site serine residue rather than a cysteine. In contrast, F. gigantica cathepsin 84 does possess a cysteine in the active site. Interestingly, our PCR expression profiling data indicated that FhCB4 is expressed in both the infective life stages of the parasite and in the adult worms. To investigate this further we set out to elucidate the activity of FhCB4 in following heterologous expression in Pichia pastoris along with a mutant enzyme in which the active site cysteine was present (FhCB4S29C). Unfortunately, none of the proteases showed functional activity against the f1uorogenic substrates Z-Arg-Arg-AMC, Z-Phe-Arg-AMC and Z-Leu-Arg-AMC, as the mature peptide failed to get auto-processed from the propeptide. The structural analysis of FhC84, FgCB4 and FhC82 using PyMOL software has shown negligible differences in the catalytic triad, questioning the proposal that it is an inactive protease.