This protocol details a highly sensitive and specific method for detecting and quantifying toxins produced by Gram-positive bacteria in food samples using enzyme-linked coagulation assay (ELCA). The present protocol provides a framework applicable to various toxins, with specific examples for staphylococcal enterotoxins (SEs) and Bacillus cereus emetic toxin (BCET). Key steps include food sample preparation, microplate coating with capture antibodies, sample and standard loading, conjugate addition, substrate reaction, and absorbance measurement. Data analysis involves constructing a standard curve and calculating toxin concentrations in samples based on established cut-off levels for safety. This protocol offers several advantages over conventional methods, including high sensitivity, rapid results, and reduced cross-reactivity. Its versatility makes it suitable for diverse foodborne toxins, contributing to enhanced food safety monitoring and quality control.