Background : The 1,2-unsaturated PAs, reported to be widely present in medicinal plants belonging to Asteraceae, Boraginaceae, and Fabaceae, cause hepatotoxicity and genotoxicity in humans and animals. Hence, there is a need for an analytical method that allows these dangerous plant toxins to be determined. In this study, we developed a method that can be used for the rapid and accurate determination of nine toxic PAs in medicinal plants using ultra-pressure liquid chromatography–electrospray ionization–quadrupole–time-of-flight mass spectrometry (UPLC-ESI-Q-TOF). Methods and Results : The compounds were eluted onto a C18 column with 0.1% formic acid and acetonitrile, and separated with good resolution within 11 min. all analytes was characterized by its precursor ions generated by ESI-Q-TOF and fragment ions produced by collision-induced dissociation (CID), which were used as a reliable database. The proposed analytical method was verified with reference to the ICH guidelines. The proposed UPLC-ESI-Q-TOF method was applied to four medicinal plants, and lycopsamin, echimidine, senkirkine and senecionine were detected by matching with reference standard, and additional six PAs were tentatively identified though chemical profiling. In addition, the QuEchERS method was the most efficient in comparison with methods like hot water and methanol in extraction efficiency of pyrrolizidine alkaloids. Conclusion : The our proposed method can determine PAs rapidly and accurately in medicinal plants and will be utilize as an important data for other researchers who need analytical information of PAs.