The Estrogen Receptors ERa and ERß bind cofactor proteins via short LXXLL motifs. The exact regulation and selectivity of these interactions remains an open question and the role of post-translational modifications (PTMs) is virtually unexplored. Here, we designed an X7–LXXLL–X7 T7 phage display library and screened this against four ER protein constructs: the ‘naked’ ERa and ERß Ligand Binding Domains (LBDs) and the tyrosine phosphorylated ERa (pY537) and ERß (pY488) LBDs. The site-selective tyrosine phosphorylated protein constructs were obtained via a protein semi-synthesis approach. Phage display screening yielded preferential sets of peptides. LXXLL peptides with a low pI/acidic C-terminus prefer binding to the naked ERß over the phosphorylated ERß analogue and ERa constructs. Peptides with a high pI/basic C-terminus show the opposite behaviour. These findings not only show regulation of the ERß–cofactor interaction via tyrosine phosphorylation, but also suggest that ERß and its tyrosine 488 phosphorylation play crucial roles in modulating interactions of coactivators to ERa since the natural Steroid Receptor Coactivators (SRCs) feature LXXLL motifs with acidic C-termini, while the repressor protein RIP140 features LXXLL motifs with basic C-termini. This insight provides explanation for ER transcriptional activity and can lead to more focussed targeting of the ER–coactivator interaction.