Livestock infection by the parasitic fluke Fasciola hepatica causes major economic losses worldwide. The excretory-secretory (ES) products produced by F. hepatica are key players in understanding the host-parasite interaction and offer targets for chemo- and immunotherapy. For the first time, subproteomics has been used to compare ES products produced by adult F. hepatica in vivo, within ovine host bile, with classical ex host in vitro ES methods. Only cathepsin L proteases from F. hepatica were identified in our ovine host bile preparations. Several host proteins were also identified including albumin and enolase with host trypsin inhibitor complex identified as a potential biomarker for F. hepatica infection. Time course in vitro analysis confirmed cathepsin L proteases as the major constituents of the in vitro ES proteome. In addition, detoxification proteins (glutathione transferase and fatty acid-binding protein), actin, and the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase were all identified in vitro. Western blotting of in vitro and in vivo ES proteins showed only cathepsin L proteases were recognized by serum pooled from F. hepatica-infected animals. Other liver fluke proteins released during in vitro culture may be released into the host bile environment via natural shedding of the adult fluke tegument. These proteins may not have been detected during our in vivo analysis because of an increased bile turnover rate and may not be recognized by pooled liver fluke infection sera as they are only produced in adults. This study highlights the difficulties identifying authentic ES proteins ex host, and further confirms the potential of the cathepsin L proteases as therapy candidates.