Infrared (IR) spectra were measured as a function of time after dissolving human serum albumin (HSA) into D2O to investigate the secondary structure and the kinetics of hydrogen-deuterium (H/D) exchange. Two-dimensional (2D) IR correlation spectroscopy and principal component analysis (PCA) were used to analyze the obtained spectra. Two-dimensional IR spectra in the amide I and amide (I land II ') regions were generated from time-dependent spectral variations in different exposure time domains of HSA in the D2O solution. The synchronous and asynchronous spectra in each time domain provide a clear separation of amide bands due to the different secondary structures and the asynchronous spectra show the specific sequence of the secondary structure exposed to the H/D exchange. PCA was used to select the appropriate time domains for the calculation of 2D correlation spectra. It was found that the loadings plots of PCA are useful also to assist the band assignments in the amide I and II region and to investigate the mechanism of the H/D exchange. The present study demonstrates the potential of PCA-2D correlation analysis combined method in the efficient separation of component bands due to different secondary structures and in the study of the kinetics of H/D exchange reaction.