Background Pyrrolizidine alkaloids (PAs) are naturally occurring plant toxins associated with potential hepatic and carcinogenic diseases in humans and animals. The concern of PAs has been increased as the consumption of herbal medicines is increased. Objective This study aimed to develop and validate a sensitive analytical method to determine 28 PAs in 5 herbal medicines using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Additionally, this study identified and quantified the amount of PA in 10 samples of each herbal medicine. Methods The pretreatment in the proposed LC-MS/MS analysis comprised solvent extraction using 0.05M H2SO4 in 50% methanol and clean-up step by MCX-solid-phase extraction (SPE) cartridge. The PA contents in herbal medicines were measured by using the developed method. Results The proposed method had recoveries ranged from 72.5% ∼ 123.7% for the Atractylodis Rhizoma Alba, 70.6% ∼ 151.7% for Alba Chrysanthmi Flos, 80.6% ∼ 130.9% for Leonuri Herba, 70.3% ∼ 122.9% for Gastrodiae Rhizoma, and 67.1% ∼ 106.9% for Glycyrrhizae Radix. Even though a few samples showed recoveries in unsatisfactory values, the proposed method indicated entirely sufficient recoveries and precisions in most samples. In monitoring results, only Leonuri Herba contained two PAs, which indicated Retrorsine (4/10) of 84.7 ∼ 120.9 μg/kg and Senkirkine (10/10) of 60.9 ∼ 170.7 μg/kg. Conclusions and Highlights As the proposed method is revealed to be a reliable procedure for monitoring 28 PAs in herbal medicines, it seems to contribute to controlling the risks coming from pyrrolizidine alkaloids in certain medicinal plants and dietary supplements.