<p>Necroptosis has been definitively confirmed as a caspase-deficient, non-apoptotic cellular mechanism that exhibits a profound connection to inflammatory disorders. The receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL Cys86) have been recognized as three main targets for necroptosis for many years. Here, we report HSPBP1 Cys201 and MLKL Cys184 as new cellular targets for necroptosis in human cells. Parthenolide, a natural sesquiterpene lactone, was first confirmed to have anti-necroptotic activity and effectively alleviated the necroptosis-induced systemic inflammatory response syndrome and abdominal aortic aneurysm (AAA) in mice. In the elastase-induced mouse AAA model, MLKL deficiency is highlighted as attenuating AAA formation. HSPBP1 Cys201 was identified to be an upstream target contributing to the anti-necroptotic activity. Co-incubated with purified HSPBP1, followed by mass spectrometry analysis, confirmed that PTL binds to HSPBP1 at Cys201, while HSPBP1 knockdown conferred a certain degree of resilience to necroptosis. Human MLKL Cys184 was discovered as another novel anti-necroptotic target in human HT-29 cells. The human MTRP and molecular dynamics results suggested that Cys184 is the potential binding site between PTL and MLKL. Our co-incubation experiments of PTL with MLKL further demonstrated that PTL can interact with the sulfhydryl group of MLKL Cys184 via covalent modification. These findings yield important insights into the complex regulatory mechanisms of necroptosis and, concurrently, underscore the therapeutic potential of PTL and its derivatives for treating AAA.</p>