The transition of drug discovery from serendipitous observation to a precise, interdisciplinary science has been propelled by the need to elucidate molecular interactions underlying disease mechanisms and pharmacological action. Although early biochemical assays and in vivo models established a foundational framework, they often lacked the sensitivity and resolution required to capture subtle binding dynamics or atomic-level structural information. The introduction of biophysical techniques represented a transformative advance, enabling real-time, quantitative, and label-free analysis of molecular interactions (Renaud et al., 2016). Pioneering methods such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy laid the groundwork for structural biology and facilitated structure-based drug design. Subsequent developments—including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and microscale thermophoresis (MST)—refined the quantification of binding kinetics and thermodynamics, significantly enhancing the precision of biomolecular interaction studies. Further innovations, such as hydrogen–deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), have addressed persistent challenges in probing protein dynamics and interaction networks. Meanwhile, thermal shift assays (TSA) and cellular thermal shift assays (CETSA) help bridge in vitro observations with cellular contexts, strengthening the translational relevance of mechanistic insights.

In this review, we provide a systematic overview of pivotal biophysical technologies that underpin modern drug discovery, including Förster resonance energy transfer (FRET), fluorescence polarization (FP), TSA/CETSA, SPR, bio-layer interferometry (BLI), MST, ITC, NMR, X-ray crystallography, and advanced mass spectrometry (MS)-based approaches such as HDX-MS and XL-MS. We outline the historical development and fundamental principles of these techniques, highlighting key innovations that have cemented their essential role in pharmaceutical research. Then, an objective assessment of each method’s strengths, limitations, and optimal application scenarios is provided. Finally, we discuss emerging trends and future directions, including improvements in detection sensitivity, automation, and high-throughput implementations. These advancements are poised to reinforce the critical role of biophysical methodologies in biomedical research and therapeutic development, particularly in the face of increasingly complex targets and the growing demands of precision medicine.