Applied Biophysics for Drug Discovery is a guide to new techniques and approaches to identifying and characterizing small molecules in early drug discovery. Biophysical methods are reasserting their utility in drug discovery and through a combination of the rise of fragment-based drug discovery and an increased focus on more nuanced characterisation of small molecule binding, these methods are playing an increasing role in discovery campaigns. This text emphasizes practical considerations for selecting and deploying core biophysical method, including but not limited to ITC, SPR, and both ligand-detected and protein-detected NMR. Topics covered include: • Design considerations in biophysical-based lead screening • Thermodynamic characterization of protein-compound interactions • Characterizing targets and screening reagents with HDX-MS • Microscale thermophoresis methods (MST) • Screening with Weak Affinity Chromatography • Methods to assess compound residence time • 1D-NMR methods for hit identification • Protein-based NMR methods for SAR development • Industry case studies integrating multiple biophysical methods This text is ideal for academic investigators and industry scientists planning hit characterization campaigns or designing and optimizing screening strategies. This chapter describes the use of MicroScale Thermophoresis (MST) for the analysis and quantification of biomolecular interactions and shows how the unique properties of MST can be used to rapidly generate binding data with high-precision and information content while minimizing time and sample consumption. It also discusses application examples, MST-specific benefits, and the integration of MST into fully automated workflows. MST was early recognized as a valuable asset to the family of biophysical approaches utilized in drug discovery research. Especially the short measurement times, versatility, easy handling, and low sample consumption, in combination with the aforementioned benefits, made MST a low-risk technique, which could be easily integrated into existing workflows without excessive investment of time and lab space. Assay development for MST-based single-point screening campaigns is performed to identify optimal buffer conditions and MST measurement parameters.