Nicotiana benthamiana is extensively used as a model herbaceous plant for virus-host interactions and sytems biology research. Here we describe a methodology to phenotype the root system architecture of N. benthamiana plants following infection with grapevine fanleaf virus, an economically important soil borne virus that is present in most vineyards worldwide. Upon completion of this protocol, images of whole root crowns can be analyzed with root phenotyping software. Following image acquisition, root tissue can be further processed for cross sectioning and ribonucleic acid extraction. Additional data (plant height, dry biomass, and leaf symptoms) were collected for correlation analysis. Documentation of root system architecture is an important first step to understand how a virus can manipulate its plant host below ground when most research is focusing on disease symptomology in leaves and fruits. This protocol allows for utilization of other viruses that infect N. benthamiana and adaptation to other plant hosts.