During the ionization process of electrospray ionization mass spectrometry (ESI-MS) in protein-ligand interaction studies, non-specific binding events may occur. The goal of this thesis was to assess the influence of non-specific binding on the apparent binding or dissociation constants Kd determined by ESI-MS and to test correction procedures for obtaining the specific dissociation constants. ESI-MS allows Kd determinations without the need of any molecular label or immobilization. Non-specific interactions may either cause false positive signals or exaggerated intensities for signals from protein-ligand complexes. Klassen [Klassen , J., Wang , W., Kitova , E., & Sun , J. (2006). Method for Distinguishing Specific from Nonspecific Protein-Ligand Complexes in Nanoelectrospray Ionization Mass Spectrometry. Anal. Chem. , 78, 3010-3018.] suggested the introduction of a reference protein, to determine the non-specifically bound fraction. In this work, several potential reference molecules (proteins and ligands) are tested to assess whether size, shape, charge, or conformational state of the molecule, affect the non-specific binding. In addition, the corrected Kd for lysozyme (Lyz) binding triacetylchitotriose (C3) is determined both by using a reference protein, cytochrome C (CytC), or a reference ligand, maltotriose (M3). The results reported here, suggest that the reference molecule, ligand or protein, should be of similar size, charge, and conformation as the respective target molecule. The determined Kd for Lyz-C3, corrected for non-specific binding with both references is in good agreement with values previously determined by other methods.