Herein, we introduce a comprehensive analytical method for the separation and relative quantification of polyglycerophospholipids (PGPLs), including phosphatidylglycerol (PG), bis(monoacylglycero)phosphate (BMP), bis(diacylglycero)phosphate (BDP), Hemi BDP, cardiolipin (CL), monolysocardiolipin (MLCL), and dilysocardiolipin (DLCL), using isotope-labeled methylation (ILM) with nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUHPLC-ESI-MS/MS). Abnormal levels of BMP and CL have been associated with the pathology of lysosomal storage and neurodegenerative diseases. Thus, simultaneous analysis of all PGPLs is important to understand the mechanisms and pathologies of such diseases. In this study, improved separation and MS detection of PGPLs, including their regioisomers, was achieved by the methylation of PGPL. ILM-based relative quantification was applied to lipid extracts from a dopaminergic cell line (SH-SY5Y) treated with drugs commonly used for Parkinson's disease (PD), resulting in the identification of 229 unique PGPLs, including 121 CLs, 71 MLCLs, and 16 Hemi BDP species. The drug treatment induced significant increases in the amount of CLs containing polyunsaturated fatty acyl chains, including 20:4 and 22:6, as well as decreased levels of BMP, Hemi BDP, and BDP species, demonstrating the feasibility of using ILM for the comprehensive and high-speed relative quantification of PGPLs.