Sap-sucking insects, such as aphids, leafhoppers, thrips, and whiteflies, are vectors for > 70% of plant viruses. Presently, the development of insecticide resistance is causing a problem in agriculture worldwide as control of these insect vectors and of virus transmission is increasingly difficult. Insects lack an adaptive immune response system but use RNA interference (RNAi) functions as antiviral defense systems. Nevertheless, some viruses that infect insects encode proteins termed as viral suppressors of RNA silencing (VSR) proteins that can act against the RNAi systems. VSR proteins enable viruses to keep and increase in number in the body of the insects. The present study aimed to construct a VSR detection system for use in the detection of VSRs of viruses transmitted by insect vectors. The activities of VSR proteins were measured using a dual-luciferase reporter assay in S2 Drosophila cells. The analyses showed that HC-pro protein from Zucchini yellow mosaic virus had the highest level of activity among the tested VSR proteins. This methodological approach enables the detection of VSR proteins and measurement of VSR activities of insect viruses as well as plant viruses transmitted by insect vectors.