Avian Influenza virus H5N1 2.3.4.4.b has recently been detected in cattle, with milk from infected animals reported to contain a high viral load, serving as a potential source for shedding and dissemination of this virus. Currently, pasteurization is the only widely recognized method for on-farm inactivation of H5N1 in milk. A current concern is that according to USDA data, less than 50% of large dairy farms pasteurize non-saleable milk, with a much lower percentage occurring in medium and small dairy farms. The objective of this pilot study was to evaluate the effect of milk acidification to a pH of ～4.0 to 4.4 and lactoperoxidase system (LPS) on the inactivation of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI) in raw whole milk. Initial trials in our study used the LPAI H6N2 virus as a surrogate for HPAI H5N1. For the milk acidification trials, citric acid was used to acidify milk. For evaluation of milk acidification and LPS, milk samples were inoculated with LPAI H6N2, with samples collected before and after treatment at various times. Evaluation of virus viability was conducted using specific-pathogen-free (SPF) embryonated chicken eggs and viral quantification using real-time quantitative PCR (RT-qPCR). Three acidification experiments were conducted using milk spiked with LPAI H6N2. Given the positive outcome observed in the inactivation of LPAI with citric acid, a fourth trial was conducted with milk containing a high load of H5N1 originating from actively infected cows. Our findings observed that MILK ACIDIFICATION with a pH between 4.1 and 4.2 resulted in the inactivation of LPAI H6N2 and HPAI H5N1 virus in milk after 6 h of treatment. Milk treatment with LPS was not effective for the inactivation of the H6N2 virus, and no further trials were conducted for this treatment option. This is the first study reporting the effectiveness of MILK ACIDIFICATION for the inactivation of HPAI H5N1 in milk originating from animals infected with H5N1. Milk acidification is an effective, accessible, and easy-to-use alternative to milk pasteurization, and future studies should evaluate the on-farm effectiveness of acidification of non-saleable milk to inactivate H5N1.