A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in dairy cows was first reported on March 25, 2024, in the United States (US), marking the first discovery of HPAIV H5N1 in cattle. Soon after, a dairy worker on an affected dairy farm became the first human case linked directly to this outbreak. Studies with influenza A virus (IAV) require secondary methods to detect the virus in infected cells or animal models of infection. We modified the non-structural (NS) genome segment of the human A/Texas/37/2024 (HPhTX) H5N1 virus to create a recombinant virus expressing nanoluciferase (HPhTX NSs-Nluc), enabling the tracking of virus in cultured cells and mice via in vitro, ex vivo, and in vivo imaging systems (IVIS). In vitro, HPhTX NSs-Nluc showed growth and plaque characteristics similar to its wild-type (WT) counterpart. In vivo, HPhTX NSs-Nluc allowed tracking viral infection in the entire animals and in the organs of infected animals using in vivo and ex vivo IVIS, respectively. Importantly, the morbidity, mortality, and replication titers of HPhTX NSs-Nluc were comparable to those of the WT HPhTX. In vitro, HPhTX NSs-Nluc was inhibited by Baloxavir acid (BXA) to levels observed with WT HPhTX. We also demonstrate the feasibility of using HPhTX NSs-Nluc to evaluate the antiviral activity of BXA in vivo. Our findings support that HPhTX NSs-Nluc represents an excellent tool for tracking viral infections, including the identification of prophylactics or therapeutics for the treatment of the HPAIV H5N1 responsible of the outbreak in dairy cows.