Corex Periplocae (the root of Periploca sepium Bge) has been widely used in clinics. Periplocin, as one of the components of cardiac glycosides in Corex Periplocae, easily triggers cardiotoxicity when used improperly. To evaluate the toxicity of Periplocin, we used UPLC-Q-TOF/MS to investigate metabolic profiles on neonatal rat cardiomyocytes exposed to high and low doses of Periplocin (0.2 mmol/L, 0.4 mmol/L). Finally, we identified 11 biomarkers associated with toxicity through multivariate statistical analysis. A “supervised” Support Vector Machine (SVM) study was used to optimize and verify the reliability of these biomarkers. In these biomarkers, all biomarkers, including carnitine, acetylcarnitine, lysoPC(16:0), proline, glutamic acid, pyroglutamic acid, leucine, pantothenic acid, tryptophan, indoleacrylic acid and citric acid, revealed a downward trend with the increase of dosage. Moreover, pathway analysis showed that these metabolites were associated with amino acid metabolism, energy metabolism and sphingolipid metabolism, which contributes to a further understanding of the toxicity mechanism of Corex Periplocae and its clinical safety. Additionally, we demonstrate that an UPLC-Q-TOF/MS-based metabolomic approach is a powerful tool and provides a promising approach for assessing the toxicity of traditional Chinese medicine and drug safety screening.