The use of small molecules to modulate cellular processes is a powerful approach to investigate gene function as a complement to genetic approaches. The discovery and characterization of compounds that modulate translation initiation, the rate-limiting step of protein synthesis, is important both to provide tool compounds to explore this fundamental biological process and to further evaluate protein synthesis as a therapeutic target. While most messenger ribonucleic acids (mRNAs) recruit ribosomes via their 5' cap, some viral and cellular mRNAs initiate protein synthesis via an alternative "cap-independent" mechanism utilizing internal ribosome entry sites (IRES) elements, which are complex mRNA secondary structures, localized within the 5' nontranslated region of the mRNA upstream of the AUG start codon. This report describes the design of a functional, high throughput screen of small molecules miniaturized into a 1,536-well format and performed using the luciferase reporter gene under control of the viral Cardiovirus encephalomyocarditis virus (EMCV) IRES element to identify nontoxic compounds modulating translation initiated from the EMCV IRES. One activating compound, validated in a dose response manner, has previously been shown to bind the glucocorticoid receptor (GR). Subsequent testing of additional GR modulators further supported this as the possible mechanism of action. Detailed characterization of this compound activity supported the notion that this was due to an effect at the level of translation.