Cluster formation in the alternating electric field during differential ion mobility is critical for separation selectivity and is governed by two factors. One is the reduced mass, and the other factor is cluster binding energy between an ion and a neutral solvent molecule (modifier). Therefore, separations of isomeric analytes using a modifier can be related to the thermochemistry of the cluster formation, as subtle changes in the molecular structure will affect its charge delocalization and the binding energy with the corresponding modifier will be different. We have examined the relationship between calculated Gibbs free energies of the cluster formation and experimental ion mobility measurements (CoV dispersion plots) considering the most prominent ion-modifier interactions: charge-dipole, dipole-dipole, and charge-quadrupole. To explain selectivity effects due to the modifier, we have selected a series of positional isomers of sulfonamide drugs that were analyzed in positive and negative electrospray and the diastereoisomers ephedrine and pseudoephedrine in positive mode. The following modifiers were investigated: water, linear and branched alcohols, acetonitrile, acetone, toluene, and ethyl acetate. We could demonstrate a dependence of the separation selectivity of the differential mobility on the reduced mass and Gibbs free energy of the cluster formation. These results are supported by thermochemistry calculations (DFT) and interpreted by molecular modeling. Finally, we describe differential mobility spectrometry selectivity tuning for the multidimensional LCxDMS-MS separation of sulfonamide isomers in human plasma.