T-cell acute lymphoblastic leukaemia (T-ALL) is a malignant haematological cancer which poses significant therapeutic challenges due to a high level of refractory and relapsed cases and an overall low survival rate (< 50% in adults).1,2 Ferroptosis, a regulated, non-apoptotic cell death mechanism driven by iron-mediated lipid peroxidation in cells’ membranes, has emerged as a potential therapeutic strategy for T-ALL. Investigations show that specific T-cell types have impaired regulation of radical oxygen species (ROS) production, making them more susceptible to ferroptosis. This mechanism could be harnessed by artificially increasing ROS levels to target and eliminate refractory T-cells. However, the direct impact of ferroptosis inducers on the cell membranes is not fully understood and has never been characterized at nanoscale.3 To explore cells’ membrane ferroptosis-induced modifications, novel analytical techniques enabling chemical characterization at the nanoscale are required. Thus, we propose to use atomic force microscopy coupled with infrared (IR) spectroscopy (AFM-IR)4 - a technique allowing chemical identification with a spatial resolution of about 20 nm – to track the membrane modifications induced by peroxidation processes. A robust and reproducible AFM-IR protocol (sample preparation and AFM-IR settings) was developed to detect and image lipid peroxidation at the nanoscale in two T-ALL cell lines, Loucy and Jurkat, exposed to two ferroptosis inducers called RSL3 and APR-246. As a result of high-resolution AFM-IR chemical imaging, peroxidized phospholipid hotspots were identified on cells’ plasma membranes, while local spectra at specific positions exhibited IR absorption bands associated with different oxidized phospholipid end products. Jurkat cells responded strongly to ferroptosis induction, with lipid peroxidation primarily localized in the plasma membrane and pseudopodia regions. In contrast, Loucy cells exhibited a less pronounced lipid peroxidation response. This work highlights the potential of AFM-IR to investigate cells’ membrane biochemical degradation during ferroptosis in T-ALL. A better understanding of the degradation processes provides valuable insights into ferroptosis mechanisms and could contribute to a better perception of future therapeutic strategies targeting lipid peroxidation in cancer cells. (1) Raetz, E. A. and Teachey D. T., Hematology 2016 (2) Cordo’, V. et al., Blood Cancer Discov 2021 (3) Jiang, X. et al. Nat Rev Mol Cell Biol 2021 (4) Dazzi, A. et al., Journal of Applied Physics 2010