A method of prederivatization and HPLC with UV detection was developed for the simultaneous analysis of five major steroidal alkaloids of the Fritillaria species: verticine, verticinone, isoverticine, ebeiedine, and ebeiedinone. Derivatization was carried out by esterification of the hydroxyl-containing Fritillaria alkaloids to the corresponding naphthoates with 1-naphthoyl chloride. Reaction conditions were optimized and the yields of the derivatization were between 94 and 100% for all test alkaloids. Derivatized alkaloids were characterized by mass spectrometry and HPLC-MS. The validated HPLC-UV method demonstrated linear UV response at the sampling ranges used, and a test limit of 1 mu g/mL was determined for all analytes. This analytical method is simple, convenient, and readily reproducible. The developed method was applied to the analysis of the major pharmacologically active alkaloids in three medicinally used Fritillaria species: F. cirrhosa F. thunberghii, and F. hupehensis. Five major Fritillaria alkaloids were simultaneously analyzed qualitatively and quantitatively from crude extracts of each of these herbs.