AbstractIntroductionTrillium govanianum (Nag Chhatri and Teen Patra) is traditionally used for curing joint pains, wounds, and sexual disorders. Steroidal saponins are the main active components of this species. However, only a small amount of information is available about steroidal saponins of this plant.ObjectiveTo develop an ultra‐high‐performance liquid chromatography‐quadrupole time of flight tandem mass spectrometry (UHPLC‐QTOF‐MS/MS) and ultra‐high‐performance liquid chromatography‐evaporative light scattering detector (UHPLC‐ELSD) methods for the qualitative and quantitative determination of steroidal saponins in T. govanianum.MethodThe dried rhizomes of T. govanianum (100 mg) were extracted with ethanol–water (80:20, 10 mL) by ultrasonic treatment for 30 min at 40°C. The prepared sample was analysed by UHPLC‐QTOF‐MS/MS and UHPLC‐ELSD for the qualitative and quantitative determination of steroidal saponins.ResultA total of 24 saponins were identified using UHPLC‐QTOF‐MS/MS; seven of them were characterised by comparing with standards. Furthermore, five saponins [govanoside B (2), protodioscin (6), pennogenin tetraglycosides (11), borassoside E (21) and borassoside D (24)] were quantified using UHPLC‐ELSD method in different extracts and fractions of T. govanianum. The method showed good linearity (R2 ≥ 0.993), limit of detection (0.92–4.09 μg/mL), limit of quantification (3.1–13.5 μg/mL), precision [intra‐day relative standard deviations (RSDs) < 4.3% and inter‐day RSDs < 5.5%], and accuracy (84.0–110.3%). This is the first report on the quantification of 2, 6, 11, 21 and 24 in T. govanianum.ConclusionThe present study provides an efficient analytical method for the identification and quantification of steroidal saponins and will be helpful for the quality evaluation of T. govanianum.