Based on the pathogenicity in chickens, most H1-H16 avian influenza (AI) viruses cause mild diseases, whereas some of the H5 and H7 AI viruses cause systemic disease. The number of basic amino acids in the hemagglutinin (HA) cleavage site of AI viruses plays a critical role in pathogenicity. As we gain a greater understanding of the molecular mechanisms of pathogenicity, genome sequencing of the HA0 cleavage site has assumed a greater role in assessment of the potential pathogenicity of H5 and H7 viruses. We validated the use of HA cleavage site motif analysis by comparing molecular pathotyping data against experimental in vivo (IVPI and lethality) data for determination of both low pathogenicity and high pathogenicity AI virus declaration with the goal of expediting pathotype confirmation and further reducing the reliance on in vivo testing. Our data provide statistical support to the continued use of molecular determination of pathotype for AI viruses based on the HA cleavage site sequence in absence of an in vivo study determination. This approach not only expedites the declaration process of HPAIV but also reduces the need for experimental in vivo testing of H5 and H7 viruses.