A high-throughput antiviral agent screening system called the fluorescence-based local lesion inspection assay (FLLIA) has been developed. In this assay, leaves were mechanically inoculated with an engineered virus expressing a fluorescent protein. One half of each leaf was treated with a test chemical and the other half was treated with a buffer solution. The number and size of viral infection lesions were then imaged and quantified. A reduction in the number of viral infection lesions and/or decreased fluorescence intensity in the chemically treated group suggests a replication inhibition activity, while a reduction in the size of fluorescent lesions indicates a movement inhibition activity. This simple method can efficiently identify antiviral agents and primarily differentiate their action mechanisms. A step-by-step protocol of FLLIA was provided in this chapter.