Cereulide is an emetic toxin produced by some strains of Bacillus cereus. This bacterial toxin, a cyclic 1.2 kDa dodecadepsipeptide, is stable to heat and acids and causes nausea and vomiting when ingested via contaminated food. This work aimed to develop and validate a targeted analytical method applying liquid chromatography-tandem mass spectrometry (LC–MS/MS) to quantify this toxin in food and human faeces. Samples were extracted with acetonitrile in the presence of ¹³C₆-cereulide, a labelled internal standard, and purified by centrifugation and filtration. The limits of quantification were 0.5 and 0.3 µg kg⁻¹ for food and faeces, respectively. The linearity of the method was very good, with calculated R² values above 0.995. The mean recovery of the method was within the acceptable range of 70.0%–120.0%, the repeatability was not higher than 7.3%, and the highest intra-laboratory reproducibility was 8.9%. The estimated range for the expanded measurement uncertainty was between 5.1% and 18.0%. The LC–MS/MS method was used to analyse one food sample (rice) from a Belgian foodborne outbreak and five faecal samples from patients with clinical symptoms after consumption of the contaminated rice. The levels of cereulide were 12.22 µg g⁻¹ for food and between 6.32 and 773.37 ng g⁻¹ for faecal samples.