Profiling allergens in complex food ingredients used in oral food challenges and immunotherapy is crucial for regulatory acceptance. Mass spectrometry based analysis employing data-independent acquisition coupled with ion mobility mass spectrometry-mass spectrometry (DIA-IM-MS) was used to investigate the allergen composition of raw peanuts and roasted peanut flour ingredients used in challenge meals. This comprehensive qualitative and quantitative analysis using label-free approaches identified and quantified 123 unique protein accessions. Semiquantitative analysis indicated that allergens Ara h 1 and Ara h 3 were the most abundant proteins and present in approximately equal amounts and were extracted in reduced amounts from roasted peanut flours. The clinically significant allergens Ara h 2 and 6 were less abundant, but relative quantification was unaffected by roasting. Ara h 5 was undetectable in any peanut sample, while the Bet v 1 homologue Ara h 8 and the lipid transfer protein allergen, Ara h 9, were detected in low abundance. The oleosin allergens, Ara h 10 and 11, were moderately abundant in the raw peanuts but were 100-fold less abundant in the defatted roasted peanut flour than the major allergens Ara h 1, 3, 2, and 6. Certain isoforms of the major allergens dominated the profile. The relative quantitation of the major peanut allergens showed little variation between different batches of roasted peanut flour. These data will support future development of targeted approaches for absolute quantification of peanut allergens which can be applied to both food ingredients used in clinical studies and extracts used for skin testing and to identify trace levels of allergens in foods.