Cereulide is the heat-stable toxin produced by certain strains of Bacillus cereus. It is the main virulence factor of emetic B. cereus strains, which causes the emetic food poisoning syndrome, including rare fatal cases of food intoxications. Due to presumably low intoxication doses, a sensitive, specific, and robust technique is needed for its detection. In 2002, a LC-MS method was developed which allowed absolute quantification of cereulide using valinomycin as standard. This study describes the validation,according to the Commission Decision 2002/657/EC, of the LC-MS² method, a tandem mass spectrometry technique, which guarantees lower detection limit and higherspecificity. The LC-MS² method, calibrated with valinomycin, was validated in rice and tested on various matrices (i.e., red beans, spices, and chili con carne) containing cereulide. The process combines a simple extraction step from the food matrix followed by LC-MS² analysis and detection by ion trap mass spectrometer. The detection limit for cereulide in rice was 0.5 ng eq/g, which is 20 to 2,500 times lower than currently understood intoxicative doses between 10 and 1.280 ng/g previously reported for cereulide. The validated method was specific, sensitive, repeatable, and reproducible with recoveries ranging from 77% to 101%.