The signaling pathway of transforming growth factor-beta (TGFβ) plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small molecule inhibitors specifically targeting SMAD4, the downstream master regulator of TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGF-β signaling. In this study, we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format. This assay enables simultaneous monitoring of the protein-protein interaction (PPI) between SMAD4 and SMAD3, as well as the protein-DNA interaction (PDI) between SMADs and their consensus DNA binding motif. The multiplexed TR-FRET assay exhibits high sensitivity, allowing the dynamic analysis of the SMAD4-SMAD3-DNA complex at single amino acid resolution. Moreover, the multiplexed uHTS assay demonstrates robustness for screening small molecule inhibitors. Through a pilot screening of an FDA-approved and bioactive compound library, we identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFβ signaling agents.