We determined crucial parameters, such as delipidation efficiency, system robustness, and back-exchange levels, and minimized unspecific protein/peptide adsorption to the column matrix. We also investigated titanium oxide (TiO 2 ) beads as an alternative to ZrO 2 -based protein delipidation. Furthermore, we established an automated wash method for bead regeneration in parallel to peptide LC-MS analysis, which enables intervention-free sample acquisition and makes the phospholipid trap column reusable. Finally, we demonstrated the functionality of the delipidation protocol by HDX-MS analysis of empty and loaded membrane scaffold protein phospholipid nanodiscs, the latter loaded examples containing multidrug efflux pump transmembrane subunit AcrB. We envision that this automated and robust delipidation workflow will make HDX-MS analysis of membrane-embedded proteins routine.