Five herpesvirus have been reported to infect horses and 3 belong to the Alphaherpesvirinae subfamily (equine herpesvirus-1, EHV-4 and EHV-3). Among them, EHV-1 is the most pathogenic and causes respiratory disease in young horses, abortion in mares, neonatal death and neurologic damages, also call equine herpesvirus myeloencephalopathy. EHV-4, also named the rhinopneumonia virus, is closely related to EHV-1 but mostly causes respiratory disease and only sporadic abortion and neonatal infection in horses. EHV-3 is distinct from previous viruses and is responsible for equine coital exanthema, characterized by formation of papules, pustules, ulcers and vesicles on the external genitalia of horses. The equine industry economical loss linked to EHV infection is significant, which warrants surveillance and prophylaxis. Currently, no antiviral molecule has a marketing authorization for equine species despite critically needs, warranting the development or adaptation of drugs that are effective against equine viruses.The aims of this work were 1) to investigate the efficacy of Real-Time Cell Analysis (RTCA) system to monitor EHV infections in equine dermal cells 2) to assess the effectiveness of acyclovir (ACV) and ganciclovir (GCV) against EHV-1, EHV-4 and EHV-3 by RTCA system and 3) to investigate the capacity of RTCA system for the screening of compounds library in order to identified new inhibitors of these viruses. To confirm results achieved, monitoring of cell morphology by microscopy and quantification of viral loads were perform in parallel.Antiviral effect of ACV or GCV against the EHV-1 KyD strain was evaluated by RTCA. Results showed that ACV and GCV prevented the impedance decrease induced by EHV-1 infection in a dose-dependent manner and EC50 measured were 9.88 ± 2.14 µg/ml and 0.62 ± 0.49 µg/ml, respectively. The ACV and GCV EC50 against EHV-4 were 17.38 ± 5.95 µg/ml and 2.79 ± 0.25 µg/ml, respectively. Against EHV-3, the ACV and GCV EC50 were 15.23 ± 2.85 µg/ml and 1.86 ± 0.46 µg/ml, respectively. All the data were confirm by qPCR and microscopy observation. The screening of compounds library allowed identifying several molecules with an antiviral activity needing further investigation.This study confirms the efficiency of RTCA to monitor cytopathic effect formation induced by equine alphaherpesviruses on E. Derm cells in real-time. In this model, GCV was shown to be the most effective against all viruses used to infect E. Derm cells. This technology aims to complement and support conventional methods used in the field of virology.