Infrared spectroscopy and Raman spectroscopy, when applied to food samples, have the unique ability to interrogate their chemical structure. In this research, Raman and mid-infrared (MIR) spectroscopy in conjunction with selected chemometrics approaches were used to investigate the effects of ageing on the protein structure in Cheddar cheese. The results illustrate the potential of Raman confocal imaging and MIR spectroscopy for an understanding of chemical changes in complex dairy matrices.