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The identification of small molecule inhibitors of plant virus RNA replication is useful to develop antiviral drugs and to dissect various steps in the replication process. Moreover, small molecule inhibitors could be effective tools to study similarities in replication strategies of various RNA viruses. By using Tomato bushy stunt virus (TBSV), we tested the effect of various inhibitors of host factors known to facilitate TBSV replication as well as acridine and phenanthridine derivatives, which are known anti-prion compounds, on TBSV replication. In this chapter, the methodology used to identify the direct targets of the chemicals during the different steps of replication of TBSV is described.