Recent rapid growth of lipidomics is mainly attributed to technological advances in mass spectrometry. Development of soft ionization techniques, in combination with computational tools, has spurred subsequent development of various methods for lipid analysis. However, none of these existing approaches can cover major cellular lipids in a single run. Here we demonstrate that a single method of liquid chromatography coupled with mass spectrometry (LCMS) can be used for simultaneous profiling of major cellular lipids including glycerophospholipids (PLs), sphingolipids (SPLs), waxes, sterols (ST) and mono-, di- as well as triacylglycerides (MAG, DAG, TAG). We applied this approach to analyze these lipids in various organisms including Saccharomyces cerevisiae and Schizosaccharomyces pombe. While phospholipids and triacylglycerides of S. pombe mainly contain 18:1 fatty acyls, those of S. cerevisiae contain 16:1, 16:0 and 18:1 fatty acyls. S. cerevisiae and S. pombe contain distinct sphingolipid profiles. S. cerevisiae has abundant inositol phytoceramides (IPC), while S. pombe contains high levels of free phytoceramides as well as short chain phytoceramides (t18:1/20:0-B) and IPC (t18:1/20:0-B). In S. cerevisiae, our results demonstrated accumulation of ergosterol esters in tgl1Δ cells and accumulation of various TAG species in tgl3Δ cells, which are consistent with the function of the respective enzymes. Furthermore, we, for the first time, systematically characterized lipids in S. pombe and measured their dynamic changes in Δplh1Δdga1 cells at different growth phases. We further discussed dynamic changes of phospholipids, sphingolipids and neutral lipids in the progress of programmed cell death in Δplh1Δdga1 cells of S. pombe.