In this study, we employed a gel-free proteomic approach to investigate proteome changes in both LM1 and IPEC-J2 cells after co-incubation. QExactive Orbitrap mass spectrometry (MS) was used for a full proteomic scan of bacterial and intestinal cells, and for a large-scale quantitative analysis of protein dynamics during host-microbe interaction. This is the first proteomic study to use this method to obtain novel insights into probiotic adhesion to IECs.