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Red ginseng is a steamed and dried processing product of the cultivating root and rhizome of Panax ginseng C. A. Mey., which is one of the most widely used in ginseng products. The main active ingredient of red ginseng is ginsenoside, which is different from the content of ginseng caused by thermal process, such as chemical transformation by hydrolysis reaction and hydrated reaction. In recent years, the treatment of type 2 diabetes mellitus (T2DM) by red ginseng has attracted extensive attention. Ginsenoside has the effect of treating T2DM, but it is not easy to be absorbed directly by oral administration, and the bioavailability is low. Therefore, the metabolism of ginsenoside of red ginseng was studied in this research. The influence of intestinal flora on diseases has been paid more and more attention. Studies have shown that drug molecules can interact with intestinal microorganisms to produce metabolites of intestinal flora and active components of traditional Chinese medicine transformed by intestinal flora, which may be a new way to reveal the potential mechanism of action of red ginseng. Therefore, it is necessary to identify and elucidate ginsenosides in feces. A method of ultra performance liquid chromatography mass spectrometry (UPLC-MS) was established for qualitative and quantitative analysis of ginsenosides in red ginseng, and the content changes of ginsenosides in feces and serum of rats after intragastric administration of total ginsenosides were analyzed. Thermo Syncronis C18 column (100 mm×2.1 mm×1.7 μm) was used, mobile phase A was 0.1% formic acid in water, mobile phase B was acetonitrile, and flow rate was 0.2 mL/min, injection volume was 5 μL, UPLC was equipped with binary linear gradient pump, mass spectrometry was adopted electrospray negative ion mode. Ginsenosides were identified by the information of the retention times, molecular mass, and specific MS/MS fragments. The developed UPLC-MS method for quantitative analysis of ginsenosides was validated with respect to linearity, precision, repeatability, stability, and recovery. The result showed that the detection method of ginsenoside is speedy and accurate. Ginsenosides NotoR1, Rg1, Re, Rf, Rg2, Rh1, Rb1, Rc, Ro, Rb2, Rb3, Rd, Rg3, Rk1 in feces and serum of rats are detected, which show that the intestinal flora did not completely transform ginsenoside, and a part of ginsenosides are absorbed into the blood as prototype components playing a role in the treatment of T2DM. The changes of metabolites of ginsenoside in feces were studied with the prolongation of intervention time, which provided a certain basis for the pharmacodynamics of ginsenosides in red ginseng.