Electrospray ionization mass spectrometry has recently become the technique of choice for rapid characterization of lignin degradation products. However, the fundamental question of the relationship between lignin structure and ionization efficiency has not been explored. In this work, we studied the electrospray ionization response of five structurally similar β-O-4' model lignin compounds using lithium cationization in the positive electrospray ionization mode. The studied compounds have the same β-O-4' backbone structure but differ at the α-position by increasing nonpolar side chains. Our results show a correlation between the ionization response and the length of the nonpolar side chain, with analytes having the longest side chain recording the highest ESI response in the full scan mode. Factors affecting the formation of analyte ions and analyte cluster ions were also studied. We have shown for the first time in this work that the introduction of a nonpolar group onto a β-O-4' lignin compound can increase the lithium cationization ESI response in the positive ion mode.