Giardia duodenalis is a protozoan parasite and major contributor worldwide of diarrhoea and gastroenteritis in vertebrates, including humans. Giardiasis is characterised by proliferation of trophozoites in the small intestines, where parasites attach to the gut mucosa via a ventral disk resulting in infection pathology. Our experiment quantifies protein changes in trophozoites exposed to host soluble factors (HSF) from HT-29 intestinal epithelial cells (IEC), compared to the co-incubation with the cell-monolayer itself. This host-cell co-incubation permits attachment of trophozoites to host cells. All treatments, including the control, were incubated in serum-free DMEM for 6 hours at 37°C in 5% CO₂. Two complementary quantitative approaches; Tandem Mass Tags (TMT) labelling and label-free based on spectral counting approach were performed to quantify the differentially expressed proteins of biological triplicates between conditions. We identified a total of 1650 proteins, of which a small subset of 21 up-regulated and 14 down-regulated proteins were reproducibly differentially expressed, including up-regulation of known and putative virulence factors. Cathepsin B precursor, a confirmed immunomodulatory protein, was up-regulated in trophozoites exposed to HSF, but was not changed during co-incubation with host cells. Similarly, VSP antigenic switching was increased in trophozoites incubated with HSF compared to co-incubation. Several signalling proteins were up-regulated, in particular the Tenascins, during incubation with HSF. These results indicate that induction of virulence factors from several important protein families occurs independent of trophozoite attachment to host cells. Significantly, we observed equivalent up-regulation, in both HSF-exposed and co-incubated trophozoites, of the Giardia duodenalis cystatin homologue and protease inhibitor, which may represent a novel pathogenicity factor in Giardia.