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<i>Gymnadenia conopsea</i> R. Br. is a traditional Tibetan medicinal plant that grows at altitudes above 3000 m, which is used to treat neurasthenia, asthma, coughs, and chronic hepatitis. However, a comprehensive configuration of the chemical profile of this plant has not been reported because of the complexity of its chemical constituents. In this study, a rapid and precise method based on ultra-high performance liquid chromatography (UPLC) combined with an Orbitrap mass spectrometer (UPLC&#8722;Orbitrap&#8722;MS/MS) was established in both positive- and negative-ion modes to rapidly identify various chemical components in the tubers of <i>G. conopsea</i> for the first time. Finally, a total of 91 compounds, including 17 succinic acid ester glycosides, 9 stilbenes, 6 phenanthrenes, 19 alkaloids, 11 terpenoids and steroids, 20 phenolic acid derivatives, and 9 others, were identified in the tubers of <i>G. conopsea</i> based on the accurate mass within 3 ppm error. Furthermore, many alkaloids, phenolic acid derivates, and terpenes were reported from <i>G. conopsea</i> for the first time. This rapid method provides an important scientific basis for further study on the cultivation, clinical application, and functional food of <i>G. conopsea.</i>