The toxicity assessment of herbal medicines is important for human health and appropriate utilization of these medicines. However, challenges have to be overcome because of the complexity of coexisting multiple components in herbal medicines and the highly interconnected organismal system. In this study, a target profiling approach was established by combining the characteristic fingerprint analysis of herbal chemicals with potential toxicity through a precursor ion scan-based mass spectroscopy and the target profiling analysis of biomarkers responsible for the toxicity. Through this newly developed approach, the comparative hepatotoxicity assessment of two herbal medicines from the same genus, Senecio vulgaris L. and Senecio scandens Buch.-Ham, was performed. Significant differences were found between the two species in their chemical markers (i.e., pyrrolizidine alkaloids) and biomarkers (i.e., bile acids) responsible for their toxicities. This result was consistent with the conventional toxicity assessment conducted by histopathological examination and clinical serum index assay on experimental animal models. In conclusion, this study provided a new approach for the hepatotoxicity assessment of herbal medicines containing pyrrolizidine alkaloids, which are widely distributed in various herbal medicines. The target profiling approach may shed light on the toxicity assessment of other herbal medicines with potential toxicity.