A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in the United States dairy cattle was first reported on March 2024, followed by a zoonotic cattle-to-human virus transmission to a dairy farm worker in Texas. To facilitate real-time tracking of HPAIV H5N1, we generated a recombinant Nluc-expressing H5N1 virus, HPhTX NSs-Nluc, by introducing a nanoluciferase (Nluc) reporter into the non-structural gene of human A/Texas/37/2024 H5N1 (HPhTX). HPhTX NSs-Nluc replicated with kinetics and plaque morphology comparable to wild-type virus in vitro. In vivo and ex vivo, HPhTX NSs-Nluc allowed tracking viral infection in the living animals and their necropsied organs using in vivo imaging systems (IVIS). Treatment with Baloxavir effectively inhibited HPhTX NSs-Nluc replication, comparable to wild-type virus, validating its applicability for high-throughput screening of potential antiviral therapeutics. These results demonstrate that HPhTX NSs-Nluc is a robust tool for studying H5N1 pathogenesis and assessing antiviral efficacy against HPAIV H5N1.