In this study, an ELISA-based method was observed to overestimate quantification of peanut in three wheat flour-based dry matrices (both raw and cooked). A reliable method using liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed to quantify peanut accurately in these matrices. This method simplified both sample preparation and quantification compared with previous approaches, by digesting peanut proteins without reduction and alkylation and using Ara h 1 to build calibration curves. A significantly increased amount of peanut peptides was detected by heating the extracts. The sensitivity and linearity were superior to most commercial ELISA kits. The limit of quantification (LOQ) was 0.31 mg/kg total peanut in wheat flour and the linear range was between 0.3 and 40 mg/kg. This LC-MS/MS method could be applied as a confirmatory method for peanut allergen management when positive ELISA results raise suspicion of contamination.