We developed a “non-denaturing, high-resolution structural method” using low-temperature plasma (LTP)–generated hydroxyl radicals coupled with liquid chromatography–mass spectrometry (LC-MS) to probe conformational changes during protein liquid–liquid phase separation (LLPS). Using Tau protein as a model, we found that this method reveals specific solvent accessibility changes and global conformational unfolding in the terminal regions on both sides of the protein during LLPS without chemical additives or denaturation. This method overcomes incompatibilities between LLPS systems and MS, enabling residue-level structural analysis of dynamic, disordered proteins linked to neurodegenerative diseases.