Cationic polymers can bind DNA to form polyplexes, which are noncovalent complexes used for gene delivery into the targeted cells. For more insight on such biologically relevant systems, the noncovalent complexes between the cationic polymer poly(ethylene imine) (PEI) and the nucleotide mimicking dye Cibacron Blue F3G-A (CB) were investigated using mass spectrometry methods. Two PEIs of low molecular weight were utilized (M-n approximate to 423 and 600 Da). The different types of CB anions produced by Na+/H+ exchanges on the three sulfonic acid groups of CB and their dehydrated counterparts were responsible for complex formation with PEI. The CB anions underwent noncovalent complex formation with protonated, but not with sodiated PEI. A higher proportion of cyclic oligomers were detected in PEI423 than PEI600, but both architectures formed association products with CB. Tandem mass spectrometry studies revealed a significantly stronger noncovalent interaction between PEI and dehydrated CB than between PEI and intact CB. Copyright (C) 2014 John Wiley & Sons, Ltd.