Polyethylene glycol (PEG) coatings have been widely applied in pharmaceutical and biomedical systems to prevent nonspecific protein absorption, increase vesicle blood circulation time, and sustain drug release. This study systematically investigated the planar interfacial organization of phospholipid monolayers containing various amounts of PEG conjugations before and after enzyme-catalyzed degradation of the lipids using X-ray reflectivity and grazing incidence X-ray diffraction techniques. Results showed that attaching PEG to the headgroup of the lipids up to 15 mol % had limited effects on molecular packing of the lipid monolayers in the condensed phase at the gas-liquid interface and negligible effects on the enzyme adsorption to the interface. After enzyme-catalyzed degradation, equimolar fatty acids and lyso PC were generated. The fatty acids together with the subphase Ca2+ self-assembled into highly organized multilayer domains at the interface. The X-ray measurements unambiguously revealed that the densely packed PEG markedly hindered microphase separation and formation of the palmitic acid-Ca2+ complexes.