Updated project metadata. Gram-negative bacterial infections are causing increasing levels of morbidity due to the rise of resistance to established antibiotics. New antibiotic classes with distinct molecular mode of action are therefore required. Recently, a family of macrocyclic peptidomimetics was discovered that target the outer membrane LPS transport protein LptD to specifically inhibit bacterial growth in Pseudomonas spp. To characterize the interaction of these antibiotics with LptD from P. aeruginosa, we combined photo-crosslinkable peptidomimetics and hypothesis-free mass spectrometry-based proteomics. We provide evidence that the antibiotic cross-links to the periplasmic segment of LptD, containing a ß-jellyroll domain and an N-terminal insert domain that is characteristic of Pseudomonas spp. Binding of the antibiotic to the periplasmic segment of LptD is expected to block LPS transport, consistent with the proposed mode of action and observed specificity of these antibiotics. These insights may prove valuable for the discovery of new antibiotics targeting the LPS transport pathway in other Gram-negative bacteria.