The yeast one-hybrid (Y1H) assay is a powerful molecular biology tool for studying protein-DNA interactions. This technique involves using a single DNA bait cloned upstream of a reporter gene in a yeast plasmid to determine whether a specific transcription factor (TF) or other DNA-binding protein (referred to as “prey”) can bind to the DNA bait. Typically, this technique employs a complete cDNA library to obtain multiple prey proteins, which are then introduced into yeast cells containing the bait construct. In this protocol, we present a simplified method not centered on finding new prey from cDNA libraries but on analyzing specific target plant proteins. The protocol provides a step-by-step guide for cloning DNA-bait and protein-prey constructs, transforming yeast, and screening for reporter interactions.