Abstract Cell death is a crucial physiological process. The resistance of cancer cells to therapeutic drugs is a significant barrier to successful cancer treatment and the primary factor in cancer recurrence. Activation of novel cell death pathways would resensitize drug-resistant cells to chemotherapy. Ferroptosis is a nonapoptotic cell death activated when the lipid in the cell undergoes iron-dependent peroxidation. Our overarching goal is to investigate lipid metabolism-driven cell death through the modulation of ferroptosis in cell culture and in vivo models of drug- resistant cancer. The results will provide fundamental insights into the molecular chemistry of ferroptosis and its role in disease pathology by developing a multi-omics approach with single-cell resolution. Diseases like cancer, sepsis, pre-eclampsia, diabetes, cardiovascular disease, and neurodegenerative illnesses correlate with lipids and lipid metabolism dysregulation. Lipid distributions are heterogeneous, and their chemical modifications, such as lipid peroxidation, are potentially crucial for disease onset and progression. However, the precise relationship between lipid distribution and their chemical modification and disease pathology is not fully understood. This project investigates changes in lipid distribution and lipid peroxidation both in vitro and in tissues to provide fundamental insights into their molecular chemistry and its role in disease pathology by using the experimental methods developed in our group based on spatial chemical imaging. Our approach uses Raman imaging to provide spatial information about lipids in the defined cellular compartments in contrast to the bulk or fractionated examinations of extracted lipids provided by liquid chromatography-mass spectrometry (LC-MS). This proposal builds upon our prior success in performing spatial imaging of lipid distribution in cells and tissues. The hypotheses underlying this effort are that (a) there are significant heterogeneities in lipid distributions and their chemical modifications, and (b) these heterogeneities can be correlated to the pathology of the disease. In particular, we will: 1) Investigate the effect of ferroptosis on the lipid metabolism and iron metabolism of drug- resistant cell lines in 2D culture; 2) Perform spatial mapping and profiling of lipids during ferroptosis in 3D cell culture; 3) Detect ferroptosis and identify its associated mechanism in vivo. The project outcomes will improve our understanding of the molecular mechanism, disease phenotype, and disease progression leading to better therapeutic strategies.