Human cytomegalovirus (HCMV) is a common opportunistic pathogen that can cause devastating �morbidity and mortality amongst neonates and immune-compromised patients. The current standard of care for HCMV infection is limited to four antiviral compounds that have major limitations in terms of long-�term use, toxicity, and use during pregnancy. To provide patients with alternative treatment options to decrease HCMV-related morbidity and mortality, new drugs with novel modes of action are warranted. Here, we describe a validated high-throughput fluorescence antiviral screening assay based on infection of fibroblast cells with a fluorescently tagged reference strain of HCMV (AD169-GFP) to screen and profile HCMV inhibitors.