Recent reports indicate a growing number of A/H5N1 infections in companion animals and wildlife, particularly across Europe and the United States and, most recently, in Australia, where the virus poses a potentially serious threat to endemic wildlife, including threatened species. In Poland, following an epizootic that occurred in summer 2023, A/H5N1 virus has continued to be detected in domestic animals showing clinical signs. Continued occurrence of mammalian infections remains possible, underscoring the need for reliable diagnostic approaches. Our experience suggests that A/H5N1 infection can remain challenging to diagnose even in animals with severe clinical signs and rapidly progressive, often fatal disease. In selected clinical and post-mortem samples, low viral RNA loads and/or the presence of tissue-derived PCR inhibitors can hamper amplification and subsequent sequencing. Nevertheless, by mid-2026, we had identified more than 40 A/H5N1 infections in dogs, cats, and ferrets, highlighting the continued need for sensitive molecular diagnostic approaches in mammalian cases. The optimized workflow enables robust downstream targeted Sanger sequencing of hemagglutinin (H5) and neuraminidase (N1) amplicons, providing a rapid diagnostic rescue approach for subtype confirmation and targeted genetic characterization. By refining diagnostic workflows for low-viral-load samples, this protocol enables molecular diagnostic confirmation of HPAI H5N1 subtype in companion animals.