The novel Electron-Vibration-Vibration Two-Dimensional InfraRed (EVV 2DIR) technique is a nonlinear spectroscopic method that measures the vibrational coupling spectrum in a way analogous to the measurement of spin couplings by 2D NMR methods. To date, the main focus of this work has been on the development of EVV 2DIR as a tool for high-throughput, label-free protein identification and absolute quantification. One of the most promising areas where EVV 2DIR technique can provide complementary information not available via other established proteomic methods is for the study of post-translational modifications. This spectroscopy has demonstrated absolute quantification of phosphorylation levels in peptides, something difficult to achieve with other methods. The cyclin-dependent kinase inhibitor p27 is involved in orchestrating a variety of protein interactions in vivo that are key modulators of cell-cycle progression and that are often deregulated in cancer. The regulation of p27 is controlled by phosphorylation on serine, threonine and tyrosine residues thus providing a good model for EVV 2DIR studies.