When targeting a certain class of analytes, such as the phosphorylated lipids in complex biological extracts, interfering species can pose challenges to qualitative and quantitative analyses. Two aspects of lipid analysis were optimized to simplify the isolation and characterization of phosphorylated lipids in biological extracts. A new solid ionic crystal MALDI matrix was synthesized which combined the lipid response enhancing UV-absorber p-nitroaniline with the protonating agent butyric acid. Mass spectra of the extracts containing phosphorylated lipids were simplified by revealing only protonated molecules [M + H]+ of the zwitterionic phosphatidylcholine (PC) headgroup-containing lipids, such as lyso-PC, PC, and platelet-activating factor. For the anionic phosphorylated lipids, such as phosphatidylglycerol, phosphatidic acid, and phosphatidylserine, further spectrum simplification is obtained by the appearance of only the monosodium adducts [M + Na]+ as the major molecular ions, in preference to the double sodium adducts [M + 2Na - H]+. In addition, a new extraction, isolation, and cleanup procedure has been developed to prepare the phosphorylated lipids for MALDI-TOF analysis by the use of immobilized metal ion affinity chromatography media (i.e., ZipTip). The latter procedure was successfully applied to a complex biological tear film lipid layer extract in preparation for MALDI-TOF analysis and phospholipid characterization.