Niujiaodihuang Detoxify Decoction (NDD) is an integrated traditional Chinese medicine prescription that has been used as a therapeutic agent for the treatment of acute liver failure (ALF). However, the mechanisms underlying its action remain unclear.To determine the protective effect of NDD on D-galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF and explore the underlying mechanisms.We characterized the NDD fingerprint by HPLC and established D-GalN/LPS-induced ALF models in Sprague-Dawley rats and LO2 cells. Next, we measured the protective and antiferroptotic effects of NDD in vivo and in vitro. To further investigate the molecular mechanisms underlying the effects of NDD, we performed metabolomic analysis of the liver tissue using LC-MS/MS.Results of serum biochemical analysis, liver histopathology, and cell viability showed that NDD effectively relieved the liver injury. It reduced the accumulation of labile iron and alleviated lipid peroxidation by enhancing GPX4 activity. The mitochondrial morphology indicated that NDD exerted its hepatoprotective effect through an antiferroptotic activity. Metabolomic analysis showed that NDD treatment increased the levels of cysteine, decreased those of glutamate, and ameliorated the D-GalN/LPS-induced reduction in the levels of glutathione (GSH). The results for intracellular levels of reduced (GSH) and oxidized (GSSG) glutathione were consistent with those of metabolomic analysis.Our findings indicate that NDD exerts hepatoprotective activity by evoking the reprogramming of GSH metabolism, and thereby, inhibiting ferroptosis.Copyright © 2021 Elsevier B.V. All rights reserved.