The first half of my talk will be focused on the developing 13C=16O isotope labeled amide unit as a Sum Frequency Generation vibrational probe for antimicrobial peptide ovispirin-1 at various interfaces. We have demonstrated that the linewidth of site-specific SFG vibrational probes are indicative of its microenvironment and signals of different polarization combinations reveal the site-specific orientation information of interfacial peptides. In the second half, we study the denaturant-induced (urea, methylurea, dimethylurea and tetramethylurea) dynamic changes and hydration states of CN probes incorporated into aromatic side chains of peptides using two-dimensional vibrational photon echo spectroscopy and fluorescence spectroscopy. We provide the first experimental evidence that methylated urea molecules crowd more effectively around the hydrophobic side chains compared to urea. Especially, we have shown that, tetramethylurea, the strongest denaturant in the series, preferentially shields and dehydrates the tryptophan like at a pair of “tweezers”, which is not observed for other derivatives in the series.