The effects of different dihydroxybenzoic acid (DHB) isomers, when used as matrix materials in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), were investigated in analyses of polyethylene glycol (PEG) polymers. PEG polymers ranging from 400 to 8,000 Da were prepared in different DHB isomer matrices using solvent-based and solvent-free methods. PEG samples were detected only in matrices of 2,3-DHB, 2,5-DHB, and 2,6-DHB while the most intense peaks were observed using 2,6-DHB in both solvent-free and solvent-based preparations.