In this thesis, measurements of electron-vibration-vibration two dimensional infrared (EVV 2DIR) spectra are demonstrated for the first time from aliphatic, aromatic and amide containing compounds. Bioanalytical applications of EVV 2DIR spectroscopy are explored and the first EVV 2DIR measurements on proteins reported. Operational protocols for collecting EVV 2DIR spectra are described and it is shown for the case of benzene Fermi resonances how excitation pulse ordering can be used to isolate different EVV coherence pathways, giving signals that report on several aspects of a molecule’s electrical and mechanical anharmonicity. Experimental spectra are compared with first principles simulations and in general found to be in good agreement with one another. It is shown that inter-pulse delays can substantially clean up frequency domain spectra and for the range of compounds studied, the 2DIR spectra are significantly decongested compared with their 1D infrared and Raman counterparts. EVV 2DIR coherence lifetime measurements are reported and applied to a range of simple organic compounds. Measurements of exponential dephasing, nonexponential dephasing and quantum beating are demonstrated, with the exponential decays used to accurately measure vibrational linewidths and the quantum beats to measure line splittings.