Abstract: A sensitive and reliable method for determination of tetrodotoxin (TTX) in Nassarius was established using ultra high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The sample was extracted by 1% acetic acid-methanol solution, then the pH value was adjusted to 8.5−9.0. After purification with multi-walled carbon nanotubes (MWCNTs), the sample was subjected to an analytical procedure. The mobile phase containing acetonitrile and 0.1% formic acid solution with a linear gradient elution was utilized to separate TTX on an Amide column. The quantitative analysis was carried out with an external standard method in the multiple reaction monitoring mode using positive electrospray ionization. The calibration curve for TTX was linear (R>0.999) from 0.3 ng·mL^–1 to 50 ng·mL^–1. The recoveries were 83.7%−91.4% with the RSDs of 2.3%−8.6%. The limits of detection and quantification were 0.3 μg·kg^–1 and 1 μg·kg^–1, respectively. This method is suitable for determination of TTX in Nassarius.