Somatic Y537S and D538G mutations within the estrogen receptor alpha ligand-binding domain (ERα-LBD) have been linked to enhanced cell proliferation, survival, and metastases in ER-positive breast cancers. Such mutations are thought to confer ligand-independent receptor activation by increasing the flexibility of helix 12 (H12), a segment within the ERα-LBD that acts as a dynamic regulator of ERα activity. We employed bipartite tetracysteine display coupled with the biarsenical profluorophore FlAsH-EDT