Cereulide is a toxic cyclic depsipeptide produced by certain strains of Bacillus cereus found in soil and food products. While some harmless strains of Bacillus are used as probiotic, others can cause nausea and vomiting, and represent an important food safety concern. Current detection methods are time consuming and do not necessarily detect toxic cereulide. Here, we developed a rapid protocol using Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry that detects the toxin originating from a colony smear of B. cereus. The distinct molecular feature of the toxin peak at m/z 1,191 was clearly identified from bacterial extracts with a limit of detection (LOD) of 30 ng/mL. Final optimisation of the sample preparation was based on cereulide chelating cations to produce the alkali adduct [M + K]+ without the use of a MALDI matrix, and provided a 1,000-fold improvement of LOD with 30 pg/mL of cereulide. We evaluated the application of this method for the detection of cereulide in rice, milk, and different ready-to-eat meals. The proposed protocol is quick, easy and provides an improvement over conventional methods for the detection of B. cereus toxin.