The plaque reduction assay (PRA) is the gold standard phenotypic method to determine herpes simplex virus (HSV) and human cytomegalovirus (HCMV) susceptibilities to antiviral drugs. However, this assay is subjective and labor intensive. Here, we describe a novel antiviral phenotypic method based on real-time cell analysis (RTCA) that measures electronic impedance over time. The effective drug concentrations that reduced by 50% (EC 50 s) the cytopathic effects induced by HSV-1 and HCMV were evaluated by both methods. The EC 50 s of acyclovir and foscarnet against a reference wild-type ( H erpes simplex viruses 1 (HSV-1) and 2 (HSV-2) cause orolabial and genital infections as well as keratitis, encephalitis, and neonatal infections. Human cytomegalovirus (HCMV) is responsible for mononucleosis-like syndromes and organ-specific diseases in immunocompromised patients. All antiviral agents currently approved for the treatment of HSV and HCMV infections ultimately target the viral DNA polymerase (1). First-line antiviral agents for the treatment of HSV and HCMV infections include the nucleoside analogues acyclovir (ACV) and ganciclovir (GCV), respectively. Both drugs require a first phosphorylation by the thymidine kinase (TK) encoded by the UL23 gene (HSV) or the phosphotransferase encoded by the UL97 gene (HCMV) and two subsequent phosphorylations by cellular kinases to be converted into their active forms (2-4). The triphosphate forms compete with dGTP for incorporation into replicating DNA. Acyclovir triphosphate acts as a DNA chain terminator to inhibit viral replication, whereas ganciclovir triphosphate slows down DNA polymerization and eventually stops chain elongation. The pyrophosphate analogue foscarnet (FOS) is a second-line antiviral drug for the treatment of HCMV diseases and may also be used in the treatment of infections caused by nucleoside analogue-resistant HSV mutants. Foscarnet does not require any phosphorylation to be active (5). It directly inhibits the activity of the viral DNA polymerases encoded by UL30 (HSV) and UL54 (HCMV) genes. Foscarnet binds to the pyrophosphate binding site and blocks the release of pyrophosphate from the last nucleoside triphosphate added onto the growing DNA chain. Prolonged treatment with nucleoside analogues may be required to prevent or to manage HSV/HCMV infections in the immunocompromised host. Such prolonged antiviral therapy may result in the selection of viral isolates with reduced drug susceptibilities (6, 7).The plaque reduction assay (PRA) is the gold standard phenotypic method to determine the susceptibilities of HSV isolates to antiviral drugs and is approved as a standard protocol by the Clinical and Laboratory Standards Institute (8). The PRA has also been standardized in a consensus protocol for HCMV to decrease high interassay and interlaboratory variabilities (9). In this assay, cells are infected with a constant viral inoculum. The virus is then allowed to grow in the presence of serial drug dilutions for 2 to 3 (HSV) to 7 to 8 (HCMV) days before t...