Abstract Body: Cardiac fibrosis impairs cardiac function, but no clinical therapies exist. To address this unmet need, we employed a high-throughput drug screening for antifibrotic compounds using human iPSC-derived cardiac fibroblasts (CFs) (Figure 1). Counter-screening of the initial candidates using iPSC-derived cardiomyocytes and endothelial cells excluded hits with toxicity. This screening process identified artesunate as the lead compound. Following profibrotic stimuli, artesunate inhibited proliferation, migration, gel contraction in human primary CFs, reduced collagen deposition and improved contractile function in 3D-engineered heart tissues. Artesunate also attenuated cardiac fibrosis and improved cardiac function in a heart failure mouse model. Mechanistically, artesunate molecularly targeted myeloid differentiation factor 2 (MD2) and inhibited MD2/toll-like receptor 4 (TLR4) signaling pathway, alleviating fibrotic gene expression in CFs (Figure 2). Our study is the first to use multiscale drug screening, which combines human iPSC platform, tissue engineering, animal models, in silico simulation, and multiomics to identify MD2 as a therapeutic target for cardiac fibrosis.