The prevalence of foodborne illnesses arising from Gram-positive bacterial toxins constitutes a significant public health challenge, emphasizing the imperative for expeditious and accurate detection methodologies to enable timely intervention. This book chapter explores recent strides in the application of matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF) tandem mass spectrometry for the detection of these toxins in food samples. The technique’s remarkable sensitivity, high resolution, and capacity to analyze intricate mixtures of proteins and peptides position it as a promising tool for the identification and characterization of Gram-positive bacterial toxins. In the methodology outlined, researchers extract proteins or peptides from diverse food matrices, subjecting them to MALDI-TOF-TOF analysis. This process yields mass spectra that serve as fingerprints for precise toxin identification through comparison with extensive reference databases. Beyond offering a rapid and high-throughput solution for known toxins, the versatility of this approach holds a significant potential for the discovery of novel toxin variants, enriching our understanding of bacterial pathogenicity. The book chapter underscores the transformative impact of MALDI-TOF-TOF in revolutionizing food safety practices. Its capabilities provide not only faster and more efficient identification of known toxins but also open avenues for uncovering previously unknown variants. This technological leap offers the potential for more targeted responses, significantly mitigating the risks associated with foodborne illnesses caused by Gram-positive bacteria. As food safety continues to be a critical global concern, the integration of MALDI-TOF-TOF into detection methodologies promises to play a pivotal role in safeguarding public health.