Publisher Summary The power of the Yeast Two-Hybrid (Y2H) is its ability to act as a starting point for the identification of interacting proteins. The strain MaV203 has three screening markers: GALI::lacZ, SPALIO::URA3, and GALI::HIS3. Initial screening uses the HIS3 marker to identify potential positives. Secondary screening is then done with other markers to test the strength of the interactions. Any cloning strategy can be used to generate DBORF fusion vectors. The Gateway recombination method is useful for the cloning of large numbers of DB-ORF baits. For the strains and plasmids that we describe, the DB plasmid has a LEU2 marker and the AD plasmid has a TRP1 marker, but other variations of the two-hybrid system use other markers. It is important to make sure that the selective plates one uses to match the markers of the vectors you are using. After obtaining the constructs, the next step is to introduce them into the two-hybrid strain.