Equine influenza virus (EIV) is a respiratory pathogen that causes important economic losses to the equine industry worldwide. As a result, equine influenza has benefited from several technological advances in the fields of vaccine development, diagnosis and epidemiological surveillance. A previous study demonstrated the value of Real-Time Cell Analysis (RTCA) technology for monitoring the neutralisation of H1N1 virus. This technological approach has now been applied to several equine viruses (e.g. equine herpesvirus; West Nile Virus) but has not been adapted to EIV yet. The objective of this study was to develop a RTCA model for EIV to measure 1/ neutralizing antibodies in horse serums and 2/ the antiviral activity of candidate compounds, including Zanamivir and Memantine. The RTCA model was developed on a xCELLigence platform (RTCA multiplate, Agilent®) with MDCK cells. Real-Time EIV seroNeutralisation assay (RTNA) was applied to equine serums (n = 61) with known antibody titres determined by Single Radial Haemolysis (SRH) and ranging from 0 mm² to 252 mm². The normalised Cell Index (CIn) was calculated after 30 minutes pre-incubation of EIV A/equine/Jouars/4/2006 (H3N8, Florida Clade 2 sub-lineage) strain with different equine serums and subsequent cell infection. Antiviral compounds Zanamivir and Memantine were used at concentrations ranging from 50 to 1.56 µg/mL. Experimental CIn were compared with control conditions (i.e. cell-culture with/without EIV). Our results demonstrate that the CIn decrease induced by EIV infection was significantly reduced (p < 0.05) after pre-incubation with the reference EDQM (European Directorate for the quality of Medicines and Healthcare) serum (200 mm²) and horse serums with the highest SRH titres (181 to 250 mm²). Serums with an intermediate, low or negative SRH titre (91 to 140 mm²; 27 to 82 mm² and 0 mm², respectively) did not prevent the CIn decrease induced by EIV. Besides, Zanamivir significantly reduced EIV infectivity when used at 12.5 µg/ml (p < 0.05). Memantine was not active against EIV at the concentrations used. We conclude that RTCA could be used to develop new EIV neutralization and antiviral assays and to facilitate the screening of serums and chemical libraries, respectively.