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HIGHLIGHTSNovel UPLC–MS/MS method was developed to detect 12 terpenoids simultaneously in KS‐1/2.Evaluate the toxicity of KS‐1 and KS‐2 by using the zebrafish embryo model.Useful information was provided for safe clinical application of Euphorbia kansui. ABSTRACT The dried roots of Euphorbia kansui T.N. Liou ex T.P. Wang have been traditionally used for edema in China. However, the severe toxicity caused by Euphorbia kansui has seriously restricted its clinical application. Therefore, in order to study the material basis of the toxicity attenuation effect of processing with vinegar, a rapid, sensitive, validated and reliable UPLC–MS/MS method was developed to determine twelve compounds in ethyl acetate extracts of Euphorbia kansui before and after fry‐baked with vinegar simultaneously. Meanwhile, the study of their toxic effect on zebrafish was conducted. Chromatographic separation was accomplished on Waters BEH C18 UPLC column. 0.3% formic acid in water and acetonitrile were used as mobile phase with a flow rate of 0.40mL/min and a temperature at 30°C. The analysis was performed in multiple reaction monitoring (MRM) mode using positive electrospray ionization (ESI). Furthermore, the toxic research results indicated that the toxicity of Euphorbia kansui was decreased after vinegar‐processed, which might because of the increase in the content of 5‐O‐benzoyl‐20‐deoxyingenol and the decrease in the contents of the remaining terpenoids in ethyl acetate extracts of Euphorbia kansui fry‐baked with vinegar. This study demonstrated that the method used is a powerful approach to determine the content of twelve compounds that responsible for the toxic effect of Euphorbia kansui at the same instant. And provided the experimental evidence for the rationale behind the reduction of toxicity.