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International audience; Developing reliable methodologies for detecting and quantifying allergens in processed food commodities iscrucial to support food business operators in allergen risk assessment and properly implementing precautionaryallergen labels whenever required to safeguard the health of allergic consumers. Multiple Mass Spectrometry(MS) methods have been developed so far and applied for single and multi-allergen detection in foods, generatinga heterogeneous literature on this topic, with little attention paid to the extraction and the digestion steps, crucialin delivering accurate allergen measurements.This investigation carried out within an international consortium specifically built up to convey a prototypeMS based reference method, reports on the first part of the method development, namely the optimization of thesample preparation protocol for six allergens detection (cow’s milk, hen’s egg, soy, peanut, hazelnut, andalmond) in chocolate. The latter was chosen as model complex food matrix, having a high lipid and polyphenolcontent.Different steps of the sample preparation protocol have been taken into consideration: (i) sampling, (ii)composition of the extraction buffer, (iii) protein purification, (iv) protein enzymatic digestion, (v) peptidepurification and pre-concentration, and some experiments were carried out by two independent laboratories andtwo different MS platforms to provide a first assessment of the robustness of the method under development.Fifty target peptides were monitored in multiple reaction monitoring mode and validated in different labora-tories to trace the six allergenic ingredients in the incurred chocolate and the best performing protocol for samplepreparation was identified. This work paves the way of the forthcoming full analytical validation of a prototypereference method for MS-based allergen quantification.