Laboratory identification of Candida species is often complicated by overlapping features. Species specificity is critical to the appropriate use of interventions.Accurate identification and quantification of lipid species in complex lipid mixtures are crucial for assigning biological functions to lipids of fungi. Recently, much has been achieved in the field of mass spectrometry, allowing high-throughput screening of simple and complex lipid structures. The next-generation, high-resolution mass spectrometers allow accurate analysis and a much broader spectrum for lipidomic studies; nonetheless, these are often expensive, and data analysis is complex, which presents a challenge in routine lipid studies. Alternatively, by coupling the ion trap with multiple reaction monitoring (MRM) in an HPLC-ESI-MS/MS (high-performance liquid chromatography-electrospray ionization tandem mass spectrometry) platform, we can achieve rapid, sensitive, and accurate quantification of lipids in Candida extracts. Moreover, the approach is simple and relatively cost-effective. Below, we discuss the key features of ion trap HPLC-ESI-MS/MS-based comparative lipidomics of Candida cells.