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In Vivo Assessment of Ferroptosis andFerroptotic Stress in MiceKana Ide 1 and Tomokazu Souma 1,2,31 Division of Nephrology, Department of Medicine, Duke University School of Medicine,Durham, North Carolina2 Duke Regeneration Center, Durham, North Carolina3 Corresponding author: tomokazu.souma@duke.eduFerroptosis is iron-dependent, lipid peroxidation–driven, regulated cell deaththat is triggered when cellular glutathione peroxidase 4 (GPX4)-mediated cel-lular defense is insuff i cient to prevent pathologic accumulation of toxic lipidperoxides. Ferroptosis is implicated in various human pathologies, includingneurodegeneration, chemotherapy-resistant cancers, ischemia-reperfusion in-jury, and acute and chronic kidney diseases. Despite the fact that the ferrop-totic process has been rigorously interrogated in multiple preclinical models,thelackofspecif i candreadilyavailablebiomarkerstodetectferroptosisinvivoinmousemodelsmakesitchallengingtodelineateitscontributiontokeypatho-logiceventsinvivo.Criticalstepstopracticallyevaluateferroptosisinclude,butare not limited to, detecting increased cell death and pathologic accumulationoftoxiclipidperoxidesandtestingaugmentationofobservedpathologiceventsby genetic inhibition of the glutathione-GPX4 axis or mitigation of the patho-logic process by ferroptosis inhibitors. Here, we describe methods to evaluatethese key features of the ferroptotic process in mice in vivo. Specif i cally, wedescribe methods to detect toxic lipid peroxides (4-hydroxynonenal) and celldeath (based on terminal deoxynucleotidyl transferase dUTP nick end labelingstaining) as well as a protocol to pharmacologically inhibit ferroptotic stressusing liproxstatin-1. These protocols provide tools for understanding the fer-roptotic process in mouse genetic or disease models. © 2022 Wiley PeriodicalsLLC.Basic Protocol 1: How to use liproxstatin-1Basic Protocol 2: How to evaluate ferroptosis in mouse kidneysKeywords: 4-HNEr 4-hydroxynonenal r ferroptosis r liproxstatin-1 r mousedisease modelsHow to cite this article:Ide, K., & Souma, T. (2022). In vivo assessment of ferroptosis andferroptotic stress in mice. Current Protocols, 2, e413.doi: 10.1002/cpz1.413INTRODUCTIONFerroptosis is an iron-dependent, non-apoptotic form of regulated cell death that istriggered by pathologic accumulation of toxic lipid peroxides (Bayir et al., 2020;Conrad,Lorenz,&Proneth,2021;Galluzzietal.,2018;Ideetal.,2021;Jiang,Stockwell,& Conrad, 2021; Stockwell et al., 2017). Emerging evidence suggests that ferroptosiscontributes to a wide spectrum of human diseases, such as neurodegeneration, cancers,ischemia-reperfusion (IR)-induced organ injury, stroke, traumatic brain injury, asthma,liverinjury,andacuteandchronickidneydiseases(Alimetal.,2019;Conradetal.,2021;Friedmann Angeli et al., 2014; Guan et al., 2021; Ide et al., 2021; Wenzel et al., 2017).Current Protocols e413, Volume 2Published in Wiley Online Library (wileyonlinelibrary.com).doi: 10.1002/cpz1.413© 2022 Wiley Periodicals LLCIde and Souma1 of 11