Generating DNA-bait strains for gateway-compatible yeast one-hybrid (Y1H) screens involves three steps. The first is to generate an Entry clone containing the DNA-bait of interest. Gateway cloning is used to clone larger baits, such as promoters, into pDONR-P4-P1R. (An alternative set of steps is also presented in this protocol that describes the creation of Entry clones by annealing primers and performing conventional ligation into pMW#5-a strategy best suited for smaller DNA-baits up to 100 bp.) The second is to transfer this DNA-bait from the Entry clone to the two Y1H reporter Destination vectors, pMW#2 (HIS3) and pMW#3 (LacZ). A two-step process is used because Entry clones generate a versatile resource that can be used for transfer of DNA-baits into a variety of vectors, for instance, upstream of the green fluorescent protein-encoding ORF to study spatiotemporal expression patterns. The final step is to integrate the HIS3 and LacZ reporter constructs into the genome of the Y1H yeast strain, YM4271. The entire process takes 24-32 d, plus sequence confirmation if necessary.