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Ion mobility-mass spectrometry (IM-MS) has proven to be a highly informative technique for the characterization of lipids from cells and tissues. We report the combination of hydrophilic-interaction liquid chromatography (HILIC) with traveling-wave IM-MS (TWIM-MS) for comprehensive lipidomics analysis. Main lipid categories such as glycerolipids, sphingolipids, and glycerophospholipids are separated on the basis of their lipid backbones in the IM dimension, whereas subclasses of each category are mostly separated on the basis of their headgroups in the HILIC dimension, demonstrating the orthogonality of HILIC and IM separations. Using our previously established lipid calibrants for collision cross-section (CCS) measurements in TWIM, we measured over 250 CCS values covering 12 lipid classes in positive and negative modes. The coverage of the HILIC-IM-MS method is demonstrated in the analysis of Neuro2a neuroblastoma cells exposed to benzalkonium chlorides (BACs) with C10 or C16 alkyl chains, which we have previously shown to affect gene expression related to cholesterol and lipid homeostasis. We found that BAC exposure resulted in significant changes to several lipid classes, including glycerides, sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines. Our results indicate that BAC exposure modifies lipid homeostasis in a manner that is dependent upon the length of the BAC alkyl chain.