The clinical importance of peanut (Arachis hypogaea) allergies demands standardized allergen extraction protocols. We determined the effectiveness of common extraction conditions (20 buffers, defatting reagents, extraction time/temperatures, processing, extraction repeats) on crude protein and Ara h 1 and 2 yields. Despite similar 1D-gel profiles, defatting with n-hexane resulted in significantly higher yields of crude protein, Ara h 1, and Ara h 2 than with diethyl ether. The yields were affected by the composition and pH of the extraction buffers and other conditions, but crude protein yield did not always correlate with Ara h 1 and 2 yields. Denaturants, reducing agents, acidic buffers, and thermal processing of peanuts perturbed allergen quantification in ELISAs, probably via exposure of additional epitopes. Allergen detection in 2D-Western blots with PBS resulted in greater sensitivity than with TBS or Tris. We recommend that allergen extraction conditions be selected based on the research question being investigated. (C) 2016 Elsevier Ltd. All rights reserved.