An analytical method using liquid chromatography tandem mass spectrometry has been developed for the determination of neurotoxic shellfish poisoning (NSP) toxins (brevetoxins) in shellfish. The tested parameters included extraction solvent, clean-up method, mobile phase and mass spectrometric detection parameters including multiple reaction monitoring (MRM) conditions. The toxins were extracted with 80% methanol in water and cleaned up using Strata-X SPE cartridges. Chromatographic separation of brevetoxins (PbTx-1, PbTx-2 and PbTx-3) was carried out on a C18 column (100 mm × 2.1 mm, 2.6 μm) with gradient elution. The developed method was validated according to the requirements of SANTE 11945/2015. The average recoveries of the brevetoxins ranged from 75.9% to 105.8%, the intra-day precisions were in the range of 0.4-8.6%, the inter-day precisions were in the range of 0.7–6.3% and the limits of quantification (LOQs) were 25 μg/kg for each toxin. The validated method was successfully applied to real shellfish samples, including mussel, oyster and blood cockle. Overall, the method has shown excellent sensitivity and reproducibility, and it could be used as a reference method for the monitoring of individual brevetoxins in shellfish samples.