Introduction: Roots of Gypsophila trichotoma Wend. (Caryophyllaceae) are rich sources of glucuronide oleanane‐type triterpenoid carboxylic acid 3,28‐O‐bidesmosides (GOTCABs). These saponins have been reported to possess synergistic cytotoxicity in combination with type I ribosome‐inactivating protein saporin. Objective: To develop ultra high‐performance liquid chromatography – electrospray ionisation/high resolution mass spectrometry (UHPLC‐ESI/HRMS) acquisition strategy for the recognition of Gypsophila GOTCAB saponins. Methodology: A highly‐selective hydrophilic interaction UHPLC method (Si‐HILIC UHPLC) was developed for the separation of GOTCAB saponins from the methanol‐aqueous root extract of G. trichotoma (GTR). UHPLC was coupled to an Orbitrap mass spectrometer equipped with heated electrospray ionisation (HESI) probe. ESI‐HRMS and tandem mass spectrometry (MS/MS) data of the separated compounds was used for saponins structure assignment. Results: Based on the conformity of the fragmentation of 11 previously identified GTR saponins, 21 GOTCAB forming between two and four isobaric and positional isomers are identified with proposals for their structures. Tables with assignment of characteristic fragment ions and more than 10 newly identified saponins in GTR were described. Fragmentation rules for tentative identification of three major types of saponins from GTR were summarised and possible fragmentation pathways were proposed. Type I and II consisted of acylated and sulphated GOTCABs, respectively, while type III included acylated and sulphated saponins. The type II sulphated GOTCAB saponins were all previously not described. Conclusions: The study demonstrates the potential of the coupling of highly‐selective (Si)‐HILIC UHPLC with HRMS and MS/MS detection for analysis and identification of triterpenoid saponins.