Abstract: OBJECTIVE To investigate the doxorubicin pharmacokinetics and metabolomics to give a clear idea of which substances are related with the toxicity and to find potential cardiotoxicity related biomarkers and explanation of mechanism of systematical toxicity induced by doxorubicin. METHODS Rat was treated by doxorubicin and cardiac toxicity was denoted. The pharmacokinetics of doxorubicin and doxorubicinol was study by LC-MS/MS. Rats serum and heart tissue samples were obtained and subjected to GC-MS analysis to find potential biomarkers and mechanisms of cardiotoxicity. RESULTS Pharmacokinetics study revealed the lower concentration of the secondary alcohol metabolites in serum and heart. Metabolomics analysis show that the metabolic profiling distinguished doxorubicin group from the control group and potential biomarkers such as ketone body, fatty acids and monoacylglycerols are identified. CONCLUSION Pharmacokinetics study reveal the high concentration of doxorubicin in heart with nonlinear dynamics doxorubicinol could not be the key toxicant for low concentrations in vivo in the doxorubicin's cardiotoxicity. Metabolomic analysis of serum and heart revealed the complementary evidence of alterations in fatty acid biosynthesis, lipid metabolism and ketone body metabolite after doxorubicin treatment.