Endothelial cells (ECs) play a major role in blood vessel formation and function. While chemical exposures have been shown to adversely affect EC function and blood vessel development, resulting in cardiovascular and developmental effects, large numbers of environmentally relevant chemicals have yet to be evaluated for their potential to exert vascular effects. Recent advancements in the development of stem cell technologies have resulted in the availability of human induced pluripotent stem cell (iPSC)-derived ECs that may provide a physiologically-relevant, high-throughput applicable in vitro model for toxicity screening of environmental hazards and open a possibility of testing effects across a population of individuals. In this work we aimed to develop a highthroughput screening approach, encompassing functional vascularization and cytotoxicity assays for the multidimensional evaluation of chemical effects on endothelial cell using iPSC-derived ECs and human umbilical vein endothelial cells (HUVECs). Cells were exposed to selected angiogenesis inhibitors, cytotoxic agents and vehicle in concentration-response for either 16 or 24 hours in high-throughput compatible 384-well plate format. Chemical inhibition of vascularization was quantified using EC tube formation on biological (Geltrex®) and synthetic (SP-103 Angiogenesis Hydrogel, Stem Pharm) extracellular matrices. Bioactivity was assessed by high-content live cell imaging of EC monolayers cultured on fibronectin-coated plates. Assay performance validation indicated good to excellent assay robustness, sensitivity, and replicability for both cell types based on evaluation of inter-day and -plate replicability, coefficients of variation (%CV) of vehicle controls, and Z’-factor calculation using positive and negative chemical controls. Our data demonstrate that both iPSC-derived ECs and HUVECs formed tubes on Geltrex® and Angiogenesis Hydrogel that could be affected in a concentration-dependent manner by various angiogenesis inhibitors and cytotoxic agents. In conclusion, in vitro vascularization and cytotoxicity assays were successfully established in a high-throughput compatible format that will enable evaluation of a large number of environmentally relevant chemicals for their potential effects on angiogenesis