Lanthanide(III) complexes, particularly those sensitized by ligands, are valuable for bioassays due to their unique photophysical properties, including long luminescence lifetimes, narrow emission bands, and large Stokes shifts. These properties allow for high sensitivity in fluorescence-based assays by enabling temporal resolution of lanthanide emission from background fluorescence. A major challenge is designing sensitizing ligands that provide stable, highly emissive complexes in aqueous solutions. This review discusses the development of highly luminescent Tb(III) and Eu(III) complexes for biotechnology applications, focusing on their optimization for Homogeneous Time-Resolved Fluorescence (HTRF) technology. Key strategies include the use of octadentate ligands with all-oxygen donor atoms and multi-chromophore chelates to enhance molar absorptivity. Ligands based on 2-hydroxyisophthalamide (IAM) provide high quantum yields and stability, while 1-hydroxypyridin-2-one (1,2-HOPO) ligands offer excellent photophysical properties and aqueous stability. These ligands enable efficient sensitization of Eu(III) and Tb(III) emissions, with chiral variants showing circularly polarized luminescence (CPL) activity. The photophysical properties of lanthanide complexes depend on factors such as ligand structure, symmetry, and the metal ion's coordination environment. The quantum yield and luminescence lifetime are influenced by the ligand's ability to sensitize the lanthanide emission through efficient energy transfer. TD-DFT calculations have been used to predict the absorption and emission properties of chromophores, aiding in the design of effective sensitizers. The development of Tb(III)-IAM and Eu(III)-1,2-HOPO complexes has led to significant improvements in HTRF technology, with Tb(III)-IAM complexes showing high quantum yields and stability. These complexes have been commercialized for use in HTRF assays, offering enhanced sensitivity compared to traditional Eu(III) complexes. The study highlights the importance of ligand design in achieving optimal photophysical properties for lanthanide-based bioassays.Lanthanide(III) complexes, particularly those sensitized by ligands, are valuable for bioassays due to their unique photophysical properties, including long luminescence lifetimes, narrow emission bands, and large Stokes shifts. These properties allow for high sensitivity in fluorescence-based assays by enabling temporal resolution of lanthanide emission from background fluorescence. A major challenge is designing sensitizing ligands that provide stable, highly emissive complexes in aqueous solutions. This review discusses the development of highly luminescent Tb(III) and Eu(III) complexes for biotechnology applications, focusing on their optimization for Homogeneous Time-Resolved Fluorescence (HTRF) technology. Key strategies include the use of octadentate ligands with all-oxygen donor atoms and multi-chromophore chelates to enhance molar absorptivity. Ligands based on 2-hydroxyisophthalamide (IAM) provide high quantum yields and stability, while 1-hydroxypyridin-2-one (1,2-HOPO) ligands offer excellent photophysical properties and aqueous stability. These ligands enable efficient sensitization of Eu(III) and Tb(III) emissions, with chiral variants showing circularly polarized luminescence (CPL) activity. The photophysical properties of lanthanide complexes depend on factors such as ligand structure, symmetry, and the metal ion's coordination environment. The quantum yield and luminescence lifetime are influenced by the ligand's ability to sensitize the lanthanide emission through efficient energy transfer. TD-DFT calculations have been used to predict the absorption and emission properties of chromophores, aiding in the design of effective sensitizers. The development of Tb(III)-IAM and Eu(III)-1,2-HOPO complexes has led to significant improvements in HTRF technology, with Tb(III)-IAM complexes showing high quantum yields and stability. These complexes have been commercialized for use in HTRF assays, offering enhanced sensitivity compared to traditional Eu(III) complexes. The study highlights the importance of ligand design in achieving optimal photophysical properties for lanthanide-based bioassays.