Protein-protein interactions (PPI) are critical in cellular signal transductions that play a key role in both normal and abnormal functions in cells. Therefore, modulating the activity of these interactions is a major focus in drug discovery research. In this chapter, the authors address the development, optimization and validation of HTS assays to identify small molecule modulators of PPI. They also discuss the sources of artifacts, detailed accounts of assay technologies compatible with HTS for PPI and validating the inhibition of PPI. An extensive set of references is provided, and is a must read for beginners and a review for experienced investigators.