Class O forkhead box (FOXO) transcription factors are downstream targets of the PI3K/AKT signaling pathway, which is upregulated in many tumors. AKT phosphorylates and inactivates FOXO1 by relocating it to the cytoplasm. Because FOXO1 functions as a tumor suppressor by negatively regulating cell cycle progression and cell survival, compounds that promote FOXO1 localization to the nucleus might have therapeutic value in oncology. Here the authors describe the identification of such compounds by using an image-based, high-content screen. Compounds that were active in retaining FOXO1 in the nucleus were tested to determine their pathway specificity and isoform specificity by using high-content assays for Rev and FOXO3, respectively.