The synthesis, stability, and photophysical properties of several Eu(III) complexes featuring the 1-hydroxypyridin-2-one (1,2-HOPO) chelate group in tetradentate and octadentate ligands are reported. These complexes pair highly efficient emission with exceptional stabilities (pEu ∼ 20.7-21.8) in aqueous solution at pH 7.4. Further analysis of their solution behavior has shown the observed luminescence intensity is significantly diminished below about pH ∼ 6 because of an apparent quenching mechanism involving protonation of the amine backbones. Nonetheless, under biologically relevant conditions, these complexes are promising candidates for applications in Homogeneous Time-Resolved Fluorescence (HTRF) assays and synthetic methodology to prepare derivatives with either a terminal amine or a carboxylate group suitable for bioconjugation has been developed. Lastly, we have demonstrated the use of these compounds as the energy donor in a Luminescence Resonance Energy Transfer (LRET) biological assay format.