The pace of progress in biomedical research directly depends on techniques that enable the quantitative interrogation of interactions between proteins and other biopolymers, or with their small-molecule ligands. Time-resolved Förster resonance energy transfer (TR-FRET) assay platforms offer high sensitivity and specificity. However, the paucity of accessible and biocompatible luminescent lanthanide complexes, which are essential reagents for TR-FRET-based approaches, and their poor cellular permeability have limited broader adaptation of TR-FRET beyond homogeneous and extracellular assay applications. Here, we report the development of CoraFluors, a new class of macrotricyclic terbium complexes, which are synthetically readily accessible, stable in biological media and exhibit photophysical and physicochemical properties that are desirable for biological studies. We validate the performance of CoraFluors in cell-free systems, identify cell-permeable analogs and demonstrate their utility in the quantitative domain-selective characterization of Keap1 ligands, as well as in isoform-selective target engagement profiling of HDAC1 inhibitors in live cells.