<p>Almonds and hazelnuts are widely consumed tree nuts; nevertheless, they are recognized by the U.S. Food and Drug Administration (FDA) and regulatory agencies in other countries as potential allergens requiring clear labeling on foods. While detecting these allergens during food production is essential, protein-based assays currently used to identify cross contact during food manufacturing often have variable results due to food processing effects, matrix interference, or antibody specificity. To address these challenges, the National Institute of Standards and Technology (NIST) Reference Materials (RMs) 8404 Almond Nut Flour for Allergen Detection and 8405 Hazelnut Flour for Allergen Detection were developed to harmonize detection methods. In this work, Dumas total protein assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to characterize the protein composition of the NIST RMs along with other sources of nut flours. The RMs were next evaluated using commercial enzyme-linked immunosorbent assays (ELISAs) to assess their utility in standardizing the detection and quantification of total protein from almond and hazelnut flours. From these experiments, flours processed with different manufacturing methods were found to have similar recoveries for each ELISA only when quantifying total protein rather than total nut mass. Additionally, NIST RMs 8404 and 8405 were used to improve the reproducibility of protein recovery across commercial almond and hazelnut ELISA kits. These findings provide a framework for reporting ELISA results and using RMs to facilitate more reliable and standardized allergen risk management.</p> Graphical abstract <p></p>