Gynostemma pentaphylla, one of the traditional Chinese medicinal materials, which has gradually become a research hotspot due to its rich pharmacological activities such as hypolipidemic properties, anti-inflammatory, anti-cancer and etc. The main active ingredients of Gynostemma pentaphyllum are saponins (gypenoside), flavonoids and polysaccharides. The pharmacologically active dose depends on the content of the active ingredient, so it is necessary to establish a reliable quality evaluation standard for Gynostemma pentaphyllum. However, the existing Gynostemma pentaphyllum quality control methods are not perfect, Ginsenoside Rb1 was regarded as a signature ingredient of Gynostemma pentaphylla in local standards. Surprisingly, the high-performance liquid chromatogram results displayed that the content of Ginsenoside Rb1 was not suitable as a quality marker. Therefore, a triple quadrupole time-of-flight tandem mass spectrometer method was developed to explore other potential quality markers in Gynostemma pentaphylla. As a result, a total of 24 compounds was identified tentatively. While another stable and reliable component Gypenoside XLVI was found and the content was carried out. It was expected to replace Ginsenoside Rb1 to establish a more reliable and precise quality standard of Gynostemma pentaphylla.