Highly pathogenic avian influenza (HPAI) H5N1 recently expanded its host range to include dairy cattle, and since March 2024, there have been over 1,000 cases reported in the United States. This circulation in dairy cows and resulting spillover to other mammals, including 41 documented human cases, raises the risk of reassortment and development of mutations that increase virus fitness in mammalian hosts. Research on HPAI is crucial, but due to its high pathogenicity, work with live virus must be conducted in containment level 3 laboratories. Strict biosafety procedures are essential when handling virus-containing materials to ensure safety. In this report, we explore options for inactivating the virus to enable safe work in containment level 2 (CL2) laboratories, which allow for more efficient work. We tested beta-propiolactone as an inactivation agent for HPAI in cell culture media and allantoic fluid from eggs. We also tested whether Qiagen Buffer AVL with 95% ethanol could inactivate the virus in allantoic fluid. We report that 0.1% and 0.2% beta-propiolactone and Buffer AVL with ethanol were successful in completely inactivating the HPAI in cell culture media and allantoic fluid. These inactivation methods allow safe transfer of samples to CL2 laboratories and facilitate downstream application such as vaccine testing, ELISA, qPCR, and RNA sequencing.IMPORTANCEDue to the zoonotic potential of highly pathogenic avian influenza (HPAI) H5N1 virus and its highly pathogenic features, stringent biosafety measures are essential when handling virus-containing material, and work is required to be performed in containment level 3 laboratories. If any downstream work pertaining to the inactivated virus will be performed in containment level 2 laboratories, validation of complete inactivation of the virus is required, as incomplete inactivation poses a serious risk of laboratory-acquired infections and environmental release. Our study established and validated protocols of complete inactivation of HPAI H5N1 virus in allantoic fluid as well as in tissue culture medium by beta-propiolactone and AVL plus 95% ethanol.