Histone deacetylases (HDACs), histone acetyltransferases (HATs), and the molecular chaperone heat shock protein 90 (HSP90) are attractive anticancer drug targets. Highthroughput screening plays a pivotal role in modern molecular mechanism-based drug discovery. Cell-based screens are particularly useful in that they identify compounds that are permeable and active against the selected target or pathway in a cellular context. We have previously developed time-resolved fluorescence cell immunosorbent assays (TRF-Cellisas) for compound screening and pharmacodynamic studies. These assays use a primary antibody to the single protein of interest and a matched secondary immunoglobulin labeled with an europium chelate (Eu). The availability of species-specific secondary antibodies labeled with different lanthanide chelates provides the potential for multiplexing this type of assay. The approach has been applied to the development of a 384-well duplexed cell-based screen to simultaneously detect compounds that induce the co-chaperone HSP70 as a molecular marker of potential inhibitors of HSP90 together with those that modulate cellular acetylation (i.e., potential inhibitors of histone deacetylase or histone acetyltransferase activity). The duplexed assay proved reliable in high-throughput format and f64,000 compounds were screened. Following evaluation in secondary assays, 3 of 13 hits from the HSP70 arm were confirmed. Two of these directly inhibited the intrinsic ATPase activity of HSP90 whereas the third seems to have a different mechanism of action. In the acetylation arm, two compounds increased cellular acetylation, one of which inhibited histone deacetylase activity. A third compound decreased cellular histone acetylation, potentially through a novel mechanism of action. [Mol Cancer Ther 2007;6(3):1112 -22]