Almond and macadamia nut cultivars were analyzed for physical seed characteristics, chemical composition, and protein antigenicity. The effect of environmental conditions (geographic location and harvest year) on almond seed composition was also investigated. Almond samples ranged from 0.78-1.44 g in individual seed weight, 18.3-27.8 mm in length, 9.8-13.8 mm in width, and 6.9-10.2 mm in thickness. Macadamia nut samples ranged from 2.41-3.36 g in individual seed weight, 18.7-20.1 mm in diameter, and 14.4-15.9 mm in thickness. Moisture, lipid, protein, ash, soluble sugars, and tannins ranged from 2.9-5.6%, 50.9-66.7%, 15.7-26.7%, 2.6-3.5%, 3.0-6.5%, and 0.07-0.36% in almond seeds and 1.7-2.6%, 60.0-66.2%, 5.6-7.1%, 1.0-1.4%, 5.3-8.7%, and 0.03-0.04% in macadamia nut seeds, respectively. The acidic amino acids (glutamic acid and aspartic acid) were the dominate amino acids in almond and macadamia nut proteins, accounting for 24.4-43.3% and 35.1-40.8% of total amino acids. Almond and macadamia nut seed proteins have essential-to-total amino acid ratio ranges of 25.4-33.8% and 22.5-26.1% and are also rich sources of arginine with ranges of 7.3-11.9% and 9.4-11.5%, respectively. Total free amino acids in almond seeds ranged from 172.3-723.2 mg/100 g dry weight, with free asparagine accounting for 14.9-44.9% of total free amino acids. A monoclonal-based sandwich ELISA and a polyclonal-based inhibition ELISA were developed for detecting and evaluating the antigenicity of almond and macadamia nut proteins, respectively. ELISAs, in combination with Western and Dot blot analyses, were used to investigate the antigenic stability of almond and macadamia nut proteins to various processing (thermal and pH) treatments and to quantify these proteins in complex food matrices. The ELISAs were able to detect almond major protein (AMP) and macadamia nut proteins at levels as low as 19.2 ± 1.3 ng/ml and 21.8 ± 0.9 ng/ml and were able to detect AMP and macadamia nut proteins at or below 10 ppm in majority of tested food matrices.