In yeast hybrid assays, the process of identifying preys that interact with the bait of interest involves several steps. First, in this protocol, the bait yeast strain is transformed with a library of activation domain (AD)-prey clones and plated on selective media containing 3-aminotriazole (3AT). This selects transformants containing an AD-prey clone that induces HIS3 reporter expression. Second, these "HIS-positive" colonies are analyzed for LacZ induction (and, optionally, URA3 induction in yeast two-hybrid (Y2H) assays). Third, yeast PCR is used on these "double-positive" colonies to amplify the insert from the AD-prey plasmid. Fourth, some of this PCR product is used to perform a gap-repair retest to confirm the interaction in fresh bait-strain yeast, and the remainder is used for DNA sequencing to determine prey identity for those that successfully retest. Finally, interactions are carefully examined to filter out likely false-positive interactions. This protocol takes 20-43 d plus sequence confirmation to complete.