A new LC-MS/MS method for the separation and detection of the most prominent marine lipophilic toxin groups comprising okadaic acid, dinophysistoxins, yessotoxins, azaspiracids, pectenotoxins, spirolides and some okadaic acid fatty acid esters has been developed. With this method 28 different marine lipophilic biotoxins can be analysed in a single run. Separation was achieved with an acetonitrile/water gradient containing ammonium hydroxide (pH 11). All toxins were stable under these basic conditions. Compared to chromatography using an acidic gradient, the limit of detection (LODs) for okadaic acid, yessotoxin, gymnodimine and 13-desmethyl spirolide C were improved two- to three-fold, mainly due to better peak shapes. The azaspiracids and pectenotoxins-2 showed comparable LODs under acidic and basic conditions. A major advantage of the developed method is that toxins can be clustered in retention time windows separated for positively and negatively ionized molecular ions. Therefore, there is no need for rapid polarity switching or two separate runs for one sample. The new method showed good repeatability and reproducibility and is an important step in the development of alternatives to the animal tests currently in use for shellfish toxin analysis.