Lignin is the most abundant natural resource of aromatic moieties and the second most abundant natural biopolymer. Analytical techniques that obtain as much information as possible on the exact structural content of lignin species are essential for developing efficient processes that transform highly complex lignin wastes into value chemicals and biofuels. For mass spectrometric analysis of lignin samples, usually electrospray ionization, atmospheric pressure chemical ionization, or atmospheric pressure photoionization are used as ionization techniques. Matrix-assisted laser desorption/ionization (MALDI) is less frequently applied but offers a much more rapid screening option for lignin mixtures. In this study, we compared several common MALDI matrices for analysis of alkali lignin and discovered that different chemical matrices exhibited very different ionization efficiencies and selectivity with respect to the structures of the lignin-related compounds as well as the presence of heteroatoms. Importantly, the results highlight that the choice of matrix strongly determines the analytical coverage of molecular species in the complex lignin degradation mixtures. Graphical abstract