Reactive oxygen species (ROS) are essential physiological molecules, and ROS production is strictly regulated. In many diseases, the change of physiological function and the agonist released induce the excessive increase of ROS production, and then cell function is impaired. In cardiovascular diseases, such as hypertension, diabetes, and Alzheimer's vascular lesions, the ROS production increases in vascular endothelial cells and eventually disturbs the vascular function. To better understand the role of ROS in disease, it is essential to elucidate what agonist increases the ROS production and where the ROS production occurs. Here, we show a novel high-throughput screening (HTS) protocol of what agonists induce ROS production and where it occurs in live human vascular endothelial cells using a fluorescence probe and an image cytometer. By using this HTS protocol, we found that Alzheimer’s brain-derived assemblies, amylospheroids, increase the mitochondrial ROS production because the mitochondrial ROS inhibitors (YCG-063 and mito-tempol), but not NADPH oxidase inhibitors (VAS2870 and apocynin), blocked the ROS production. By elucidating whether ROS production occurs in mitochondria or plasma membrane of live cells, this protocol will lead to a better understanding of the role of ROS in disease.