Nuclear hormone receptors (NHRs) are major regulators of development and homeostasis in multiple organ systems. These proteins are ligand-modulated transcription factors that regulate gene expression in response to changes in circulating levels of their cognate hormones or hormone analogs. When NHRs bind ligands, they adopt distinct conformations that enable or disable the binding of coregulator proteins in a manner that reflects the agonist vs. antagonist character of the ligand (1). Using the estrogen receptor ligand binding domain as a representative member of the NHR family, we show the development of functional protein microarrays and use them to explore coactivator recruitment and NHR homo-and hetero-dimer functionality. These NHR protein microarrays can be fabricated in either a forward mode (coactivator recruited to printed NHR) or a reversed mode (NHR recruited to printed coactivator). From these microarrays, we can predict the potency and pharmacological character of various NHR ligands through the nature of their coactivator recruitment. Additionally, different coactivator proteins can be functionally classified and their affinity for NHRs quantified. NHR-selective antagonist ligands and small molecule coactivator mimics disrupt the coactivator-NHR complex. This novel proteomic approach was also used to assess coactivator recruitment to explore hetero-dimer functionality. Heterodimers of the estrogen receptor were found only to recruit coactivators when both monomers are bound with agonist ligands, an observation that provides an insight into the complex biology of hormones that act on tissues containing both NHR subtypes. We can extend this NHR proteomic approach to the analysis of multidomain full-length NHR constructs and can concurrently monitor the activation state of different classes of NHRs with a mixture of endogenous or synthetic ligands of varying NHR selectivity and pharmacology.