AbstractIntroductionRhazya stricta Decne. (Apocynaceae) is a medicinal plant rich in terpenoid indole alkaloids (TIAs), some of which possess important pharmacological properties. The study material including transgenic hairy root cultures have been developed and their potential for alkaloid production are being investigated.ObjectiveIn this study, a comprehensive GC‐MS method for qualitative and quantitative analysis of alkaloids from Rhazya hairy roots was developed.MethodsThe composition of alkaloids was determined by using GC‐MS. In quantification, the ratio between alkaloid and internal standard was based on extracted ion from total ion current (TIC) analyses.ResultsThe developed method was validated. An acceptable precision with RSD ≤ 8% over a linear range of 1 to 100 µg/mL was achieved. The accuracy of the method was within 94–107%. Analysis of hairy root extracts indicated the occurrence of a total of 20 TIAs. Six of them, pleiocarpamine, fluorocarpamine, vincamine, ajmalicine and two yohimbine isomers are reported here for the first time in Rhazya. Trimethylsilyl (TMS) derivatisation of the extracts resulted in the separation of two isomers for yohimbine and also for vallesiachotamine. Clearly improved chromatographic profiles of TMS‐derivatives were observed for vincanine and for minor compounds vincamine and rhazine.ConclusionThe results show that the present GC‐MS method is reliable and well applicable for studying the variation of indole alkaloids in Rhazya samples. Copyright © 2015 John Wiley & Sons, Ltd.