Our earlier work has demonstrated that enzymatic cleavages of proteins (including tightly folded macromolecules) into smaller peptide fragments can be achieved in minutes under microwave irradiation, in contrast to hours needed by conventional methods. This novel approach has significantly accelerated the peptide mass mapping process by using mass spectrometric (MS) methods. The present work describes assessment of conformational changes in proteins upon microwave irradiation by hydrogen/deuterium (H/D) exchange experiments, followed by electrospray ionization (ESI) MS analysis. The MS data indicates that the level of deuterium exchange in proteins under microwave irradiation condition (vs. without microwave) is increased by more than 50%. However, no noticeable difference in the charge state distributions of the protein was detected in the ESI-MS. Protein-inhibitor interaction studies by H/D exchange method will also be discussed.