Doxorubicin (DOX) is a widely used chemotherapeutic agent known for its efficacy against various cancers, but its clinical application is often limited by its cardiotoxic effects. The exact mechanisms of DOX-induced cardiotoxicity remain unclear, requiring further investigation. Early diagnosis is essential to enhance the quality of life and prognosis for patients with malignancies. This study aims to identify biomarkers and therapeutic targets for DOX cardiotoxicity.Heart tissue samples from 20 DOX-treated cardiotoxic mice and 19 normal controls were analyzed using liquid chromatography-mass spectrometry (LC-MS). Multivariate statistical analysis identified differential metabolites. Key metabolites were assessed using a random forest algorithm, and ROC curves evaluated diagnostic value. H9C2 rat cardiomyoblast cells were cultured to investigate the protective effects of these metabolites.Among 291 metabolites, significant differences emerged between cardiotoxic and normal mice. Five metabolites-4-hydroxy-valeric acid, 2-methylbutanoic acid, traumatic acid, PI (18:2 (9Z, 12Z)/0:0), and MIPC (t18:0/24:0 (2OH))-showed diagnostic potential. ROC analysis indicated excellent value for 4-hydroxy-valeric acid and PI (18:2 (9Z, 12Z)/0:0) and high discriminatory power for 2-methylbutanoic acid (AUC = 0. 99). Pathway analysis highlighted glycosylphosphatidylinositol-anchor biosynthesis, unsaturated fatty acids biosynthesis, pantothenate and CoA pathways, among others, associated with DOX-induced cardiotoxicity. In addition, we found that the differential metabolite Cer (d18:0/12:0) can improve DOX-induced myocardial cell damage and inhibit apoptosis-related protein expression at the cellular level.Heart tissue metabolomics with LC-MS identified critical metabolites and pathways associated with DOX cardiotoxicity, suggesting biomarkers for early diagnosis and potential therapeutic targets to mitigate DOX-related cardiotoxicity and improve clinical outcomes.Copyright © 2025 Ding, Feng, Xu and Xu.