The goal of this research is to advance vibrational spectroscopy techniques for selective and sensitive characterization of the higher order structure (HOS) of proteins, which require specific 3D conformations to function properly. Any abnormalities in their structures can result in serious health-related problems or drug efficacy losses. However, all current methods have nontrivial limitations in obtaining reliable, rapid measurements that are sensitive to HOS. We develop advanced vibrational spectroscopic techniques that are more sensitive to structured molecular backbones than side groups. One approach is to use chiral coherent Raman spectroscopy that is sensitive to helical backbones of proteins and can acquire a spectrum >100 times faster than conventional spontaneous chiral Raman. The other approach is to use quantum cascade laser (QCL)-based absorption spectroscopy that can measure the secondary structures of proteins in aqueous solutions with a better concentration sensitivity than conventional FT-IR methods. The impacts of these advanced spectroscopic techniques are not only on biological sciences but also biopharmaceutical industries and other healthcare industries.