Amyloid fibrils are common in many diseases. This protocol uses α-synuclein fibrils, a Parkinson’s disease hallmark, to demonstrate hydrogen-deuterium exchange mass spectrometry (HDX-MS) for structural analysis. It details fibril preparation, quality assessment, HDX labeling, digestion, and mass spectrometry. Steps for peptide mapping and data interpretation are included. This workflow serves as a practical guide for researchers, particularly new comers, to study insoluble protein aggregates with distinct conformations.