Abstract Lipids fulfill an array of cellular functions by composing cellular membranes, engaging in signaling pathways, and modulating protein structure. Disruptions in carefully regulated lipid compositions perturb cellular function and correlate with cancer, neurodegeneration, diabetes, and cardiovascular disease. The correlation between aberrant lipid composition and disease has inspired lipid profiling as a powerful diagnostic tool. Analysis of structural changes that concur with disease promises to reveal highly specific biomarkers and insight into the disease development. This proposal focuses on the development of ultraviolet photodissociation (UVPD) tandem mass spectrometry approaches for the characterization of glycerophospholipids (GPL) and the cardiolipin (CL) subtype in cancerous tissue. Cancer induces a stark metabolic shift that significantly affects lipid metabolism. UVPD is a high-energy ion activation technique used for MS/MS analysis, informing subtle features that are not captured by other mass spectrometry methods. Implementing UVPD to track changes in tumorous GPL structures with fine detail offers an avenue to explore the full effects of cancer on lipid dysregulation. The objectives of this proposal include: Aim 1: Development of an LC-MS-UVPD workflow to identify changes in phospholipid structure between breast cancer tissue subtypes. A quantitative lipidomic workflow incorporating UVPD will be developed for the high-throughput analysis of GPL isomers. The proposed strategy will be applied to analyze a cohort of breast tumor lipid extracts and identify significant structural changes and prognostic biomarkers. Aim 2: Complete characterization of cardiolipins from cancer cells using UVPD. CLs are of particular relevance for cancer research but present challenging structures. UVPD is the only activation method reported to provide detailed CL characterization and will be expanded upon to characterize CLs from tumorous tissue. All together, the proposed strategies promise avenues to identify diagnostic biomarkers, potential therapeutic targets, and disease mechanisms. Importantly, these strategies would be applicable for lipidomic analyses of GPLs and CLs from other diseases.