Lipid enrichment is indispensable for enhancing the coverage of targeted molecules in mass spectrometry (MS)-based lipidomics studies. In this study, we developed a simple stepwise fractionation method using a titanium- and zirconium-dioxide-coated solid-phase extraction (SPE) silica column that separates neutral lipids, phospholipids, and other lipids, including fatty acids (FAs) and glycolipids. Chloroform was used to dissolve the lipids, and neutral lipids, including steryl esters, diacylglycerols, and triacylglycerols, were collected in the loading fraction. Second, methanol with formic acid (99:1, v/v) was used to retrieve FAs, ceramides, and glycolipids, including glycosylated ceramides and glycosylated diacylglycerols, by competing for affinity with the Lewis acid sites on the metal oxide surface. Finally, phospholipids strongly retained via chemoaffinity interactions were eluted using a solution containing 5% ammonia and high water content (45:50 v/v, 2-propanol:water), which canceled the electrostatic and chelating interactions with the SPE column. High average reproducibility of <10% and coverage of ∼100% compared to those of the non-SPE samples were demonstrated by untargeted lipidomics of human plasma and mouse brain, testis, and feces. The advantage of our procedure was showcased by characterizing minor lipid subclasses, including dihexosylceramides containing very long-chain polyunsaturated FA in the testis, monogalactosyl and digalactosyl monoacylglycerols in feces, and acetylated and glycolylated derivatives of gangliosides in the brain that were not detected using conventional solvent extraction methods. Likewise, the value of our method in biology is maximized during glycolipidome profiling in the absence of neutral lipids and phospholipids that cover more than 80% of the chromatographic peaks.