Interactions between osmolytes and hen egg white lysozyme in aqueous solutions were studied by means of FTIR spectroscopy and molecular dynamics. A combination of difference spectra method and chemometric analysis of spectroscopic data was used to determine the number of osmolyte molecules interacting with the protein, and the preferential interaction coefficient in presented systems. Both osmolytes -l-proline and trimethylamine-N-oxide (TMAO) - belong to a group of stabilizing osmolytes, and according to the preferential exclusion/hydration hypothesis, both should be excluded from the vicinity of the protein backbone and surface. We provide experimental and computational evidence that although TMAO behaves according to the hypothesis, proline does not. Our results suggest that preferential exclusion is not a universal property of stabilizing osmolytes.