Protein functions are executed through a series of structural transitions.How to probe important structural motions during protein function that are sensitive to proton transfer,electron transfer, hydrogen bonding interactions and protein folding? Here we introduce the development of infrared structural biology, an emerging technology with powerful applications for protein structural dynamics.One key element of infrared structural biology is the development of vibrational structural marker (VSM) database library that translates infrared spectroscopic signals into specific structural information.In this study, we report the development of vibrational structural markers of Tyrosine for probing hydrogen bonding interaction of tyrosine side chain buried in proteins.Quantum theory based first principle computational studies combined with isotope editing and site specific mutation of proteins were employed in this study.Infrared structural biology is expected to emerge as a powerful technique for investigating the functional mechanism of a broad range of proteins.