A high performance liquid chromatographic method for determination of tetrodotoxin (TTX) in puffer fish and shellfish was developed. Puffer ovary or other sample was extracted with 0.1% acetic acid, and defatted with dichloromethane. After being cleaned up by a Sep-pak C18 cartridge and a cation-exchange cartridge, the extract was analyzed for TTX and related substances by Nagashima's HPLC method with some modifications.Recoveries of authentic TTX added to puffer fish tissues and trumpet shell digestive gland were more than 95.0% and 80.4%, respectively. The determination limit of TTX in this method was 0.2μg/g (0.9MU/g) for puffer fish tissues and 0.5μg/g (2.3MU/g) for trumpet shell digestive gland.