A method of ultrahigh performance liquid chromatography(UPLC) with DOLPA/AOT-isooctane reverse micelles was developed for the determination of heliosupine,heliotrine,lasiocarpine and heliotridine in Cynoglossum amabile Stapf et Drumm.Experimental conditions affecting the reverse micelles such as types and concentration of surface active agent,pH value,salt species and concentration,ratio of sample and reverse micelles,extraction time and back extraction time were optimized.The optimal conditions were as follows:DOLPA /AOT concentration:06 mol/L,pH value:40,KCl concentration:02 mol/L,ratio of sample and reverse micelles:4∶1,extraction time:10 min,back extraction time:30 min.Under the optimal conditions,heliosupine，heliotrine，lasiocarpine and heliotridine were separated,and determined by UPLC with acetonitrile-02% triethylamine as mobile phase by gradient elution.Four ingredients were separated in 16 min.There existed good linear relationships for the analytes between peak areas and concentrations,with detection limits of 0030,0050，0040 and 0020 mg/L,respectively.The recoveries were in the ranges of 982%-103%,979%-102%,981%-101% and 978%-104%,respectively.The RSDs for recovery,precision and reproducibility were not more than 23%.The method is convenient，sensitive and accurate,and is suitable for the determination and confirmation of 4 alkaloid in Cynoglossum amabile Stapf et Drumm.