Cereulide-producing Bacillus cereus is the causative agent of emetic food poisoning. For epidemiological studies and control of B. cereus, rapid and simple identification of cereulide-producing strains is necessary. Recently, Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) has emerged as a vital analytical tool for microbial identification and toxin detection. This study showed that MALDI-TOF MS analysis is useful for discrimination between cereulide-producing and non-cereulide-producing strains. Colonies of 42 Bacillus cereus group strains were cultured on plate count agar, MYP agar, and NGKG agar, and subsequently subjected to MALDI-TOF MS analysis. The analysis covered the mass range of m/z 800–8,000. All 16 cereulide-producing strains determined by conventional LC-MS analysis showed a peak approximately m/z 1,191.9. This peak was assumed to be the potassium ion adduct of cereulide. The presence or absence of this peak allowed discrimination between cereulide-producing and non-cereulide-producing strains. The peak at m/z 6,263.5, originating from the ribosomal subunit protein L32, served as a control peak as an appropriate analytical procedure. Furthermore, MALDI-TOF MS analysis was applied to colonies cultured on B. cereus testing plates in milk and rice samples. MALDI-TOF MS analysis is simpler and faster than conventional methods such as LC-MS analysis. Thus, this method is a useful analytical tool for B. cereus control in industrial food production and epidemiological studies.