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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 interaction. However, existing TSAs have limitations such as time consuming, labor intensive, or low sensitivity. Here we introduce a novel acousto thermal shift assay (ATSA), the first ultrasound enabled TSA, for real-time analysis of protein thermodynamic stability. It capitalizes the novel coupling of unique acoustic mechanisms to achieve protein unfolding, concentration, and measurement on a single microfluidic chip within minutes. Compared to conventional TSA methods, our ATSA technique enabled ultra-fast (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.