Glycerolipids (GLs) are essential for cellular lipid homeostasis, while dysregulation in GL metabolism is often associated with the onset or progression of human-related metabolic diseases. The profile of GLs is thus frequently used as a molecular readout for disease phenotyping. Although mass spectrometry (MS) is the method of choice for GL profiling, the current MS methods are unable to differentiate two major types of structural isomers due to the fact that fatty acyls can be linked to different positions on the glycerol backbone (sn-positions) and the site(s) of unsaturation in acyl chains. Herein, by utilizing charge-tagging Paterno-Büchi (PB) derivatization of carbon-carbon double bond (C═C), supercritical fluid chromatography (SFC), and mobility aligned tandem mass spectrometry (MS/MS), a workflow has been developed for the sensitive and structurally informative analysis of GLs. SFC allows fast separation (within 25 min) of sn-isomers of diacylglycerols (DGs) and separation of triacylglycerols (TGs) of different chain lengths and degrees of unsaturation. Time-aligned parallel fragmentation enables multiple-stage MS/MS of the PB-derivatized lipids in a high-throughput fashion and allows pinpointing C═C location to a specific fatty acyl chain. This workflow reveals the presence of more than 500 molecular structures of neutral lipids from pooled human plasma. A comparison of human plasma samples between type 2 diabetes (N = 7) and control (N = 7) shows significant changes in isomer compositions (C18:1 Δ9 vs Δ11) from nine groups of TG and DG. These findings suggest that the developed workflow can be potentially applied to lipid marker discovery for disease monitoring or diagnosis.