High performance liquid Chromatography (HPLC) has gained importance because of versatility and easier sample preparation, greater sensitivity, and comparatively easy training of personnel. Since Reverse Phase-HPLC (RP-HPLC) is a very sensitive, specific and simple, the development of optimum RP-HPLC conditions would be valuable for future researcher in preference to relying on multistep analytical techniques to detect peanut allergens. Pastorello and Trambaioli (2001) reviewed various methods for extraction and detection of allergens from animal and vegetable foods including ion-exchange gel filtration and reversed-phase chromatography. Francisco and Resurreccion (2009) used this technique to study phenolic compounds in peanut skin extracts. Previously, Moutete et al. (1995) published the first RP-HPLC detection of peanut allergens, however the authors did not specify which peaks corresponded to the major allergens Ara h 1, Ara h 2, and Ara h 3. The goal of this study was to identify the peaks that correspond to Ara h 1, Ara h 2, and Ara h 3, as well as to standardize the method with the best resolution, wavelength and fastest elution time for their detection. A C12 and a C18 column were chosen for comparison. Both columns are known for their high protein sample recovery rate, compatibility with a range of organic solvents, and ability to function at a wide pH range (pH 1.0–10.0).