Rationale: Chrysotoxine, a bibenzyl derivative from the stems of Dendrobium medicinal herbs, has recently emerged as a promising therapeutic candidate for cervical cancer. This study aimed to characterize chrysotoxine metabolites across multiple hepatocyte species and in rat urine. Methods: Metabolites were identified and characterized using liquid chromatography coupled with benchtop Orbitrap high-resolution mass spectrometry (LC–Orbitrap–MS/MS). Structural elucidation relied on accurate mass measurements (mass error <5 ppm) and comprehensive MS 2 fragmentation pattern interpretation. Results: Twelve distinct metabolites were structurally identified. Among these, M4, M6, M7, M8, M10, M11, and M12 are newly reported. Metabolic transformations occurred via five principal pathways: hydroxylation, demethylation, glucuronidation, sulfation, and glutathione (GSH) conjugation. Cross-species analysis of hepatocytes revealed direct glucuronidation as the predominant metabolic reaction. Urinary excretion profiles in rats identified hydroxylated (M9 ) and glucuronidated (M11 ) metabolites as the major elimination products. During the metabolism, chrysotoxine can be metabolized into quinone methide and ortho quinone intermediates that can be conjugated with GSH, forming the adducts M1, M2, M3, and M5. Conclusions: This study delineates chrysotoxine metabolites in vitro and in vivo, providing critical insights for further pharmacokinetic and toxicity assessments.