One component of the safety assessment of agricultural products produced through biotechnology is evaluation of the safety of newly expressed proteins.  The ILSI International Food Biotechnology Committee has developed a scientifically based two-tiered, weight-of-evidence strategy to assess the safety of novel proteins used in the context of agricultural biotechnology.  Recommendations draw upon knowledge of the biological and chemical characteristics of proteins and testing methods for evaluating potential intrinsic hazards of chemicals.  Tier I (potential hazard identification) includes an assessment of the biological function or mode of action and intended application of the protein, history of safe use, comparison of the amino acid sequence of the protein to other proteins, as well as the biochemical and physico-chemical properties of the proteins.  Studies outlined in Tier II (hazard characterization) are conducted when the results from Tier I are not sufficient to allow a determination of safety (reasonable certainty of no harm) on a case-by-case basis.  These studies may include acute and repeated dose toxicology studies and hypothesis-based testing.  The application of these guidelines is presented using examples of transgenic proteins applied for agricultural input and output traits in genetically modified crops along with recommendations for future research considerations related to protein safety assessment.