Automation endeavors always aim for both repeatability -ideally free of user interventions -and system robustness. To meet these requirements for the automated trapping of phospholipids in HDX-MS experiments, we integrated an additional valve online with the chromatographic system but placed outside the standard two-valve Waters HDX chamber, conventionally used for standard bottom-up HDX-MS analysis (Figure ). The delipidation valve is equipped with a phospholipid trap column, which is kept refrigerated in a polystyrene box containing ice (0 °C) and supplied with eluents by an additional binary solvent manager (BSM) to provide independence from standard HDX-MS solvents. The three-valve system is of straightforward use and fully automated (in our case, controlled by the Waters MassLynx software), and can be coupled to a robot performing automated deuterium labeling and sample injection.