High content cellular imaging has increasingly met the challenges of high-throughput needs and facilitates the integration of disease relevant screens at early stages of the drug discovery process.In cancer biology the FoxO proteins are of particular interest as their activation is associated with cell cycle arrest and apoptosis and have been suggested to function as tumor suppressors.In order to identify chemical probes to study the role of FoxO proteins as well as compounds with therapeutic potential we developed a high-throughput cellular imaging assay that follows the intracelhular location of the FoxO3a protein in tumor cells.Primary image-based high-content screening of a complex library containing 34.000 distinct small chemical compounds produced 242 actives.The implementation of a hierarchical network of screens and counter-screens allowed us to pinpoint the molecular targets for many novel small molecule inhibitors.Fourteen compounds were determined to be general inlhibitors of nuclear export and not specific for FoxO3a nuclear translocation.Fifty of these compounds were found to be biochemical inhibitors of PI3Kα.Using different target deconvolution strategies including kinase profiling and cell-based profiling of changes induced by compounds with known mechanism of action we identified inhibitors of signaling effectors downstream of PI3K activity.