Oxidative stress is a crucial factor in lower success rates of clinical islet transplantation, as islets experience high levels of oxidative stress. Here, we screened FDA approved compounds for potential antioxidant effects in pseudoislets, to decrease oxidative stress and enhance pancreatic islet viability and function. Studies were performed on an in vitro “pseudoislet” spheroid, a three-dimensional culture of the INS1E beta cell line and the alphaTC1 clone 6 alpha cell line. The pseudoislets were exposed to 1280 different compounds from the Prestwick library and then subjected to oxidative stress induced by menadione and hydrogen peroxide (H2O2). Cell viability and oxidative stress levels were determined using a high-throughput fluorescence microscopy pipeline for live-cell imaging with (sub)cellular resolution. Results from our top candidate were later validated in primary human islets. Our initial screen of the 1280 compounds on retained/improved viability resulted 59 candidates, from which the top ten candidates were selected based on not having a defined antioxidant effect based on literature. These ten candidates were screened for antioxidant effects, and eight out of ten compounds were shown to efficiently reduce H2O2- and menadione-induced oxidative stress in both alpha and beta cells by 25-50%. The three most promising candidates, which did not exhibit morphological changes of the pseudoislet or changes in cell volume, were further characterized, showing that the compound sulfisoxazole was most effectively capable of reducing oxidative stress, also at short pre-incubation times. Finally, we could also validate this in primary human islets, where the compound, sulfisoxazole, also maintained control islet oxidative stress levels and islet function. We showed an effective screening strategy with 3D cell aggregates based on cell viability and cellular oxidative stress, which led to the discovery of several compounds with antioxidant capacity. Our top candidate, sulfisoxazole was effective already after 30 min pre-incubation, and effectively maintained baseline islet function, and may present a promising target to alleviate oxidative stress in pancreatic islets in e.g. clinical islet transplantation.