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Abstract  Thermal shift assays (TSAs) have been extensively used to study thermodynamics of proteins and provide an efficient means to assess protein–ligand binding or protein–protein interactions. However, existing TSAs have limitations, such as being time consuming, labor intensive, or having low sensitivity. Herein, an acousto thermal shift assay (ATSA), the first ultrasound enabled TSA, is reported for real‐time analysis of protein thermodynamic stability. It capitalizes the coupling of unique acoustic mechanisms to achieve protein unfolding, concentration, and measurement on a single microfluidic chip within minutes. Compared to conventional TSA methods, the ATSA technique enables ultrafast (at least 30 times faster), highly sensitive (7–34 folds higher), and label‐free monitoring of protein–ligand interactions and protein stability. ATSA paves new avenues for protein analysis in biology, medicine, and fast diagnosis.

On‐Chip Acousto Thermal Shift Assay for Rapid and Sensitive Assessment of Protein Thermodynamic Stability