Food poisoning caused by ingestion of Staphylococcus aureus enterotoxins is one of the most common foodborne diseases. Staphylococcus aureus is a well-studied, omnipresent bacterium which is not only found in the environment but is also part of the commensal mammalian flora. This microorganism produces enterotoxins, protein by their structure which can cause gastro-enteritis, emesis or act as superantigen. The methods used to its confirmation in food samples are mainly of microbiological character and are not quantitative. They may not be used in prevention of any foodborne outbreaks or to properly identify them. Besides, none of those methods allow unambiguous identification, nor quantification, as molecular tools are inefficient at proving the existence of the toxins in foods and immunoassays are not specific enough, not suitable for quantification and more importantly limited in the range of toxins they can identify. The proteomic approach method for the specific detection and quantification of each toxin is achieved through the analysis of peptides (toxin fragments) unique to a toxin. The peptides are obtained by extraction, purification and concentration of the enterotoxins out of the matrix and submitted to a proteotypic digestion by trypsin, The goal was to select two specific peptides per toxin to ensure proper identification and quantification. With a recognizable extraction concept the toxin is isolated from the incriminated matrix. During the process of method development the toxin was spiked at various steps down the extraction procedure to a matrix for additional control of the extraction losses. The spiked amount of toxins (1000 ng of each enterotoxin) is not representative of a real contamination but was used for the estimation. The major conclusions were that the extraction efficiency was dependent (eg milk vs meat) on the food matrix and the selection of the proteotypicpeptides