Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.