BACKGROUND Some studies have shown that Panax quinquefolium fruit (PQF) could also be used as a potential medicinal resource. However, little is known about the composition of ginsenosides and their dynamic changes at different development stages of PQF. Therefore, this study is of great significance for the metabolomics and rational utilization of PQF. METHODS The samples were analyzed using ultra-high-performance liquid chromatography combined with an Orbitrap mass spectrometer (UHPLC-Orbitrap MS), and the method of metabonomics was applied to profile the dynamic changes of ginsenosides in PQF at different development stages. RESULTS A total of 109 ginsenosides were identified or tentatively characterized. Samples collected from different development stages were significantly discriminated according to ginsenoside contents. A total of 25 potential chemical markers enabling the differentiation were discovered. CONCLUSIONS For the first time, the study developed an UHPLC-Orbitrap MS-based approach to detect ginsenoside in PQF at different development stages using a non-targeted mode. This comprehensive phytochemical profile study revealed the structural diversity and discrimination of ginsenosides in PQF at different development stages, which could provide the basis for the metabolomics and rational application of PQF.