Giardia duodenalis is the protozoan agent responsible for the majority of parasitic gastroenteritis in humans worldwide. Disease pathology includes malabsorption and maldigestion, cell apoptosis and small intestinal barrier dysfunction, which occurs in absence of known toxins, cell invasion and overt inflammation. To understand pathogenesis, host-parasite in vitro interaction models provide global insights into disease induction and virulence. Hence, we performed the first proteomic analysis of G. duodenalis trophozoites interacted with intestinal epithelial cells (IECs, HT-29) for 6 hours, and compared it to trophozoites exposed to cell-free fractions of host-soluble signals. This has allowed us to demonstrate that distinct and independent protein cascades are induced by host attachment compared to host soluble signals. We utilised a tandem mass tag (TMT) approach and evaluated it as quantitative proteomics for the first time in Giardia.