Protein-protein interactions are an integral part of virtually all aspects of cellular function. Many of these interactions are mediated by small modular units called protein interaction domains (PIDs). We do not yet understand, however, how much functional information is encoded in these modules. It has previously been shown that Nbp2SH3 and Bem1SH3b domains in S. cerevisiae bind several target peptides with the same consensus sequence, yet display finely tuned affinities for each. In this study, I have shown that there exists an evolutionarily conserved ability of orthologous fungal Nbp2SH3, Bem1SH3b, and Abp1SH3 domains to discriminate between target peptides within the same species. In addition, I have developed a method to quantitatively probe SH3 domain specificity using purified SH3 domains and naturally occurring proline-rich constructs (PRRs) in the context of cell lysate from S. cerevisiae. Expansion of this work may yield valuable insights into intrinsic SH3 domain specificity.