The central step in virus infection cycle is replication, which depends on viral and host factors. Model hosts, such as yeast, can be very valuable to identify host factors and study the functional interactions of host factors with viral proteins and/or the virus nucleic acids. The advantages of using yeast include the availability of (i) single gene-deletion library, (ii) the essential gene library (yTHC), (iii) the controllable small or large-scale expression of viral proteins and nucleic acids, and (iv) the rapid growth of yeast strains. Here, we describe procedures, which facilitate high-throughput analysis of tombusvirus replication in yeast.