A facile method based on high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC/(+)ESI-MS(n)) has been established for the analysis of polyoxypregnane glycosides in the stems of Marsdenia tenacissima. The data reveals the ability of MS(n) in the structural elucidation of polyoxypregnane glycosides including the nature of the polyoxypregnane core, the kinds of the substituents and the types of sugar residues. Offline Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) is also performed to assign accurate elemental compositions. In this study, eighteen polyoxypregnane glycosides have been investigated. Among these components, five compounds are unambiguously identified as Marsdenoside K, Tencissoside A, B, C and D; two compounds are established as novel compounds based on mass spectral data; and the other eleven compound's structures are tentatively proposed. Furthermore, breakdown curves are constructed to distinguish five pairs of isomers among these eighteen compounds. As far as our knowledge, this is the first report on identification of polyoxypregnane glycosides in the stems of M. tenacissima by HPLC/ESI-MS(n) directly, which could save time and material consuming efforts in traditional phytochemistry analyses.