Abstract: OBJECTIVE To establish a method for simultaneous determination of peimine, peiminine and peimisine in Fritillaria Thunbergii Bulbus, and compare the contents of different origin and processing methods. METHODS Hypersil C₁₈ column(250 mm×4.6 mm, 5 μm) was used, the temperature of column was maintained at 30℃, using acetonitrile(A)-0.05% triethylamine water solution (B) as the mobile phase by gradient elution at the flow rate of 1.0 mL·min^-1. Using ELSD detection and the temperatures of drift tube were maintained at 55℃, the carrier gas flow rate was 40 psi, gain was 10. RESULTS All the constituents showed good linearity(r² ≥ 0.999 6) in the range of the tested concentration. The average recoveries (n=9) of peimine, peiminine and peimisine were 100.1%, 100.1% and 96.9%, respectively. Compared the contents of different origin and processing methods, the content of peimine and peiminine in the non-sulfur-resistant group was the highest(0.082%-0.24%, average 0.15%), followed by raw sun treatment group (0.058%-0.23%, average 0.13%), followed by sulfur fumigation treatment group (0.032%-0.12%, average 0.092%), shell ash group was the lowest (0.040%-0.12%, average 0.072%). CONCLUSION The validated method has the advantages of simplicity, good precision and reliability, allowing the comprehensive quality control of Fritillaria Thunbergii Bulbus. Different processing methods have effects on the content of 3 alkaloids in Fritillaria Thunbergii Bulbus. Sulfur fumigation and shell ash treatment will reduce the content of alkaloid.