Respiratory syncytial virus (RSV) is a common virus that infects people of all ages and causes cold-like symptoms in most cases. However, more serious infections occur in the younger and older extremities of the population causing severe lung infections such as bronchiolitis and pneumonia. The current standard of care is mostly limited to supportive treatment, although prophylaxis by passive immunization with the humanized monoclonal antibody palivizumab and therapeutic intervention with aerosolized ribavirin are available. Unfortunately, administration of palivizumab is restricted to at-risk infants up to the age of two and is associated with high cost, while ribavirin treatment is hindered by questionable efficacy and safety reasons. Consequently, the development of novel specific RSV antiviral drugs is needed to help decrease RSV-related morbidity and mortality. We describe here a fluorescence-based high-throughput screening assay to discover RSV inhibitors which is based on the infection of HeLa cells with a recombinant RSV strain that contains an enhanced green fluorescent protein coding sequence in its viral genome.