The estrogen receptor alpha (ERα) features a large ligand-binding pocket capable of accommodating a variety of conformationally diverse molecules, each eliciting unique cellular responses. This structural plasticity facilitates various conformational changes, exposing different interaction surfaces for cellular proteins, triggering a myriad of biological outcomes. Alterations in the ligand-binding domain, particularly through amino acid substitutions, can modify the recognition and selectivity of ERα for agonists versus antagonists. In our study, we engineered a small library of ERα variants by modifying residues 524 and 525. These modifications resulted in variants with up to seventy-fold increased selectivity for the antagonist endoxifen and up to fifty-fold increased selectivity for the antagonist 4-hydroxytamoxifen (4-OHT) over the natural ligand estradiol. Analyzing these variants elucidates the critical roles of residues 524 and 525 in determining agonist specificity for estradiol. This advancement holds significant potential for developing selective recognition molecules, a crucial step towards creating a biosensor for endoxifen, the active metabolite used in breast cancer treatment.