Food items undergo physicochemical changes such as hydrolysis, oxidation, and rancidity, during the preparation and storage owing to their contamination with microbes. Several bacteria produce detrimental toxins that can even be lethal to the consumers. Enterotoxins, botulinum toxins, and cereulide are common toxins produced by Staphylococcus epidermidis, Clostridium botulinum, and Bacillus cereus, respectively. Recently proteomics-based techniques, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) and mass spectrometry (MS), have enabled the detection of toxins present in traces. Liquid chromatography-mass spectrometry (LC-MS) is an advanced tool which analyzes complex mixtures and is highly sensitive making it suitable for the detection of foodborne toxins. Nonetheless, its large-scale adoption for the purpose of food safety assessment still faces certain hurdles like the cost of instrument, requirement of technical expertise to handle the complexity, and analysis of data using sophisticated softwares. The constant advancements in the field of MS and its merging with complementary techniques offers a big leap forward as far as food safety is concerned. This chapter gives a detailed protocol of LC-MS to detect foodborne gram-positive bacterial toxins.