2D IR spectroscopy has been utilized to directly observe of conformational dynamics of biological systems for both equilibrium and non-equilibrium pathways. Observing specific structural changes associated of active biomolecules provides insights into the molecular movements leading to their functionality. Well-positioned probe pairs provide structural information while simultaneously detecting the dynamics in the region where biological function takes place. A systematic study of a variety of 2D IR probes, including cyano- modes, ring modes, and azido- modes will be discussed, focusing on the coupling between different pairings of these vibrational reporters as well as their sensitivity to frequency fluctuations due to their local environment