Pathologic angiogenesis has emerged as an important therapeutic target in several major diseases. Zebrafish offer the potential for high-throughput drug discovery in a whole vertebrate system. We developed the first quantitative, automated assay for antiangiogenic compound identification using zebrafish embryos. This assay uses transgenic zebrafish with fluorescent blood vessels to facilitate image analysis. We developed methods for automated drugging and imaging of zebrafish in 384-well plates and developed a custom algorithm to quantify the number of angiogenic blood vessels in zebrafish. The assay was used to screen the LOPAC1280 compound library for antiangiogenic compounds. Two known antiangiogenic compounds, SU4312 and AG1478, were identified as hits. Additionally, one compound with no previously known antiangiogenic activity, indirubin-3'-monoxime (IRO), was identified. We showed that each of the hit compounds had dose-dependent antiangiogenic activity in zebrafish. The IC(50) of SU4312, AG1478, and IRO in the zebrafish angiogenesis assay was 1.8, 8.5, and 0.31 micromol/L, respectively. IRO had the highest potency of the hit compounds. Moreover, IRO inhibited human umbilical vein endothelial cell tube formation and proliferation (IC(50) of 6.5 and 0.36 micromol/L, respectively). It is therefore the first antiangiogenic compound discovered initially in a zebrafish assay that also has demonstrable activity in human endothelial cell-based angiogenesis assays.