The introduction and subsequent detection of highly pathogenic avian influenza (HPAI) in poultry is influenced by the virus replication fitness, transmission fitness, and virulence in poultry. These viral fitness parameters are important for implementing surveillance and control measures for poultry. This study investigates the potential application of an avian in vitro model using primary chicken embryo (CEF) and duck embryo fibroblasts (DEF) to identify the viral fitness for a reference panel of eight dominant HPAI clade 2.3.4.4 virus genotypes: four H5N1 viruses isolated between 2021 and 2024, as well as three H5N8 and one H5N6 virus isolated between 2014 and 2020. Infectious virus titre and cytopathogenicity were measured in the primary cell cultures over time and these data were analysed using a mathematical model which delineates cell populations into susceptible, latent, infectious, and dead compartments. In addition to obtaining traditional virological parameters such as peak virus replication and the time to 50% cell death, eight new parameters, key among those, the infecting time (tinf), generation time (tgen) and basic reproduction number (R0), were estimated using the mathematical model. Collectively, these parameters contribute to virus characterization, enhancing the resolution for comparing genetically similar viruses. This approach can allow for the evaluation of virus virulence, replication fitness, and, ideally, transmissibility fitness across different hosts. This study underscores the potential of integrating avian in vitro models with mathematical modeling and builds towards rapid risk assessments of novel HPAI viruses.