Thyroid hormone receptors (TRs) are nuclear receptors that are activated by thyroid hormone ligands and co-regulator proteins. Two receptor subtypes, TRalpha and TRbeta, have been suggested to play a role in numerous physiological functions. However, specificity of receptor subtype function and co-regulator interaction is unclear due to the lack of TR subtype-specific ligands. Five TR ligands were evaluated for their selectivity and interaction with the TR subtypes. A multiplex assay was used to identify co-regulator peptide interaction, and biochemical assays were used to characterize ligand-receptor specificity. In the biochemical assay, rank order ligand potencies were similar in the presence of co-activator peptides, SRC1-2 and SRC3-2, and the co-repressor peptide, NCoR1-2, with T3 and Triac potencies greater in the presence of the co-repressor. The potency of Tetrac was similar regardless of the co-regulator used while T4 and rT3 demonstrated selectivity for TRalpha subtype. The rank order among TR ligands at either receptor subtype in the biochemical assay correlated with the multiplex assay. These assays can be used to identify new ligands that can provide further insight into TR biology.