Characterization of protein secondary structures at interfaces in situ and in real time is important to understand biological processes associated with cell membranes and solve problems in various fields of biomedical sciences and engineering. However, such characterization is challenging because it requires methods that are selective to both interfaces and protein secondary structures. We demonstrated that chiral sum frequency generation spectroscopy (SFG) can provide amide I and N-H stretch vibrational signals that are free of water background and characteristic to various protein secondary structures such as parallel beta-sheet, anti-parallel beta-sheet, alpha-helix, 3-10 helix and disordered structures, thus enabling characterizations of proteins at interfaces, similar to circular dichroism spectroscopy for secondary structural characterizations in solution. Using chiral SFG, we studied an amyloid protein, human islet amyloid polypeptide (hIAPP) that is associated with type II diabetes. We observed in real time conformational changes of hIAPP from disordered structures to alpha-helices and then beta-sheets upon interactions with a lipid surface. We analyzed the amide I spectra of the misfolded hIAPP in beta-sheet structures and extracted information about its orientation at the lipid surface, providing insight into pathogenic mechanism of the amyloid disease. We also used chiral N-H/N-D stretching modes as a probe for monitoring real-time proton exchange in protein backbones, introducing a method for studying dynamics and solvent exposures of proteins at interfaces. These results demonstrate chiral SFG as a new tool for characterizing protein structures at interfaces that can potentially be used to address a wide range of fundamental and engineering problems.