BACKGROUND Since March 2024, cases of highly pathogenic avian influenza (HPAI) caused by A(H5N1) virus of clade 2.3.4.4b have been reported in dairy cattle in the United States, followed by spillover to avian and other mammalian species including humans. Although human-to-human transmission has not been reported, the virus's ability to infect mammals and potential of adaptation raise public health concerns, necessitating enhanced monitoring and preparedness. AIM We aimed to develop digital RT-PCR assays to detect and quantify influenza A(H5N1) 2.3.4.4b viruses in biological and environmental samples. METHODS We developed two digital RT-PCR assays targeting the matrix protein (JRC-MP) and haemagglutinin (JRC-HA) genes of A(H5N1) 2.3.4.4b viruses. After in silico assessment of inclusivity and exclusivity, we evaluated the assays’ performance using RNAs from influenza A(H5N1) viruses isolated from infected animal specimens, in an inter-laboratory exercise with diverse target and non-target isolates, and on wastewater samples either negative or spiked with A(H5N1) 2.3.4.4b RNA. RESULTS The JRC-MP assay detects influenza A viruses of different subtypes and origins, while the JRC-HA assay specifically detects HPAI A(H5Nx) 2.3.4.4b strains. The assays demonstrated high sensitivity, showing consistent results in the inter-laboratory exercise. They also detected target RNAs in wastewater samples with high accuracy, despite background components, supporting potential use in wastewater surveillance programmes. CONCLUSIONS Aligned with One Health strategies for zoonotic avian influenza surveillance, we propose the combined use of these two assays for the rapid and sensitive detection of influenza A(H5Nx) 2.3.4.4b in biological and environmental samples to enhance monitoring and outbreak control measures.