Lactate dehydrogenases (LDHs) are tetrameric enzymes of therapeutic relevance for cancer therapy due to their important implications in cancer cell metabolism. LDH active site inhibition suffers from different drawbacks due to several features such as high cellular concentration and a shared active site among the dehydrogenase family. Conversely, targeting the LDH oligomeric state is an exciting strategy that could provide a suitable alternative to active-site inhibition. In the present study, we developed a biophysical screening cascade to probe the LDHs tetrameric interface. Using nanoscale differential fluorimetry (nanoDSF) as a primary screening method, we identified a series of hits that destabilize the tetrameric protein. From this primary screening, we validated selected hits using saturation transfer difference nuclear magnetic resonance (STD NMR) and microscale thermophoresis (MST) as a combination of orthogonal biophysical techniques. Finally, we characterized the validated hits and demonstrated that they specifically interact at the tetrameric interface of LDH-1 and LDH-5 and can inhibit the LDH tetramerization process. Overall, this work provides a convenient method for screening ligands at the LDH tetrameric interface and has identified promising hits suitable for further optimization. We believe that this biophysical screening cascade, especially the use of (nano)DSF, could be extended to other homomeric proteins.