The use of ultrahigh throughput screens (uHTS) is a well-accepted mechanism to identify agonists and antagonists of target receptors. We used the Path Hunter [Path Hunter technology is a registered trademark of DiscoveRx Corporation.] technology from DiscoveRx to screen the entire Merck compound library for glucocorticoid receptor (GR) agonists in a 2.2-microl total reaction volume assayed in a 3456-well plate format. This single addition, homogenous assay which utilizes the principle of enzyme fragment complementation (EFC) to detect nuclear translocation of GR, an initial step of receptor activation, was used to successfully screen a large library of small molecules as indicated by an average signal to background ratio of approximately 4-fold and an average Z-factor value of 0.45. Hits from the HTS campaign were studied in a cytokine secretion assay in primary human monocytes to gain functional information regarding these compounds in a phenotypic and physiologically relevant setting. Our data indicate that using the PathHunter assay, we successfully identified compounds that showed agonism for the GR receptor in primary human monocytes and due to their performance in a physiologically relevant model they likely will have a better chance to evoke clinical efficacy.