This study investigates the role of fatty acid desaturases 1 and 2 (FADS1/2) in lipid metabolism and ferroptosis susceptibility in triple-negative breast cancer (TNBC). FADS1/2 are responsible for the biosynthesis of polyunsaturated fatty acids (PUFA), which are crucial for maintaining the architecture of the plasma membrane. The study found that aggressive TNBC cells exhibit higher expression of FADS1/2, leading to increased PUFA availability and enhanced susceptibility to ferroptosis, a form of iron-dependent cell death. Lipidomic analysis and functional assays revealed that targeting FADS1/2 using genetic and pharmacological approaches rendered these tumors resistant to ferroptosis, while exogenous PUFA supplementation sensitized them to ferroptosis induction. Additionally, inhibiting lipid droplet (LD) formation and turnover suppressed the buffering capacity of LD, potentiating iron-dependent cell death. These findings were validated in vitro and in vivo using mouse and human-derived models, as well as in a retrospective cohort of TNBC patients. The study highlights a novel metabolic vulnerability in aggressive TNBC cells and suggests that targeting FADS1/2 and LD biogenesis could be a promising therapeutic approach for treating this aggressive form of breast cancer.This study investigates the role of fatty acid desaturases 1 and 2 (FADS1/2) in lipid metabolism and ferroptosis susceptibility in triple-negative breast cancer (TNBC). FADS1/2 are responsible for the biosynthesis of polyunsaturated fatty acids (PUFA), which are crucial for maintaining the architecture of the plasma membrane. The study found that aggressive TNBC cells exhibit higher expression of FADS1/2, leading to increased PUFA availability and enhanced susceptibility to ferroptosis, a form of iron-dependent cell death. Lipidomic analysis and functional assays revealed that targeting FADS1/2 using genetic and pharmacological approaches rendered these tumors resistant to ferroptosis, while exogenous PUFA supplementation sensitized them to ferroptosis induction. Additionally, inhibiting lipid droplet (LD) formation and turnover suppressed the buffering capacity of LD, potentiating iron-dependent cell death. These findings were validated in vitro and in vivo using mouse and human-derived models, as well as in a retrospective cohort of TNBC patients. The study highlights a novel metabolic vulnerability in aggressive TNBC cells and suggests that targeting FADS1/2 and LD biogenesis could be a promising therapeutic approach for treating this aggressive form of breast cancer.