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Panax quinquefolius (PQ) has been widely used in traditional Chinese medicine and functional food. Ginsenosides are the important functional components of PQ. The ginsenosides’ diversity is deeply affected by the processing conditions. The ginsenosides in the steamed PQ have been not well-characterized yet because of the complexity of their structure. In the study, the comprehensive investigation of ginsenosides was performed on the steamed PQ with different steaming times and temperatures by UPLC-Q-TOF-MS. Based on the molecular weight, retention time and characterized fragment ions, 175 ginsenosides were unambiguously identified or tentatively characterized, including 45 protopanaxatriol type, 49 protopanaxadiol type, 19 octillol type, 6 oleanolic acid type ginsenosides, and 56 other ginsenosides. Ten new ginsenosides and three new aglycones were discovered in the steamed PQ samples through searching the database of CAS SciFinderⁿ. Principal component analysis showed the significant influence on the chemical components of PQ through different processing conditions. The steaming temperature was found to promote the transformation of ginsenosides more than the steaming time. The protoginsenosides were found to transform into the rare ginsenosides by elimination reactions. The malonyl ginsenosides were degraded into acetyl ginsenosides, and then degraded into neutral ginsenosides. The sugar chain experienced degradation, with position changes and configuration inversions. Furthermore, 20 (S/R)-ginsenoside Rh1, Rh2, Rg2, and Rh12 were found to transform from the S-configuration to the R-configuration significantly. This study could present a comprehensive ginsenosides profile of PQ with different steaming conditions, and provide technical support for the development and utilization of PQ. Full article