To protect allergic patients and guarantee correct food labeling, robust, specific and sensitive detection methodsare urgently needed. Mass spectrometry (MS)-based methods could overcome the limitations of current detectiontechniques. Thefirst step in the development of an MS-based method is the identification of biomarkers, whichare, in the case of food allergens, peptides. Here, we implemented a strategy to identify the most salient peptidebiomarkers in peanuts. Processed peanut matrices were prepared and analyzed using an untargeted approach viahigh-resolution MS. More than 300 identified peptides were furtherfiltered using selection criteria to strengthenthe analytical performance of a future, routine quantitative method. The resulting 16 peptides are robust to foodprocessing, specific to peanuts, and satisfy sequence-based criteria. The aspect of multiple protein isoforms isalso considered in the selection tree, an aspect that is essential for a quantitative method’s robustness but seldom,if ever, considered.