Modifiers provide fast and reliable tuning of separation in differential mobility spectrometry (DMS). DMS selectivity for separating isomeric molecules depends on the clustering modifier concentration, which is typically 1.5–3 mol % ratio of isopropanol or ethanol in nitrogen. Low concentrations (0.1%) of isopropanol were found to improve resolution and sensitivity but at the cost of practicality and robustness. Replacing the single-channel DMS pump with a binary high-performance liquid chromatography (HPLC) pump enabled the generation of modifier mixtures at a constant flow rate using an isocratic or gradient mode, and the analytical benefits of the system were investigated considering cyclohexane, n-hexane, or n-octane as nonclustering modifiers and isopropanol or ethanol as clustering modifiers. It was found that clustering and nonclustering modifier mixtures enable optimization of selectivity, resolution, and sensitivity for different positional isomers and diastereoisomers. Data further suggested different ion separation mechanisms depending on the modifier ratios. For 85 analytes, the absolute difference in compensation voltages (CoVs) between pure nitrogen and cyclohexane at 1.5 mol % ratio was below 4 V, demonstrating its potential as a nonclustering modifier. Cyclohexane's nonclustering behavior was further supported by molecular modeling using density functional theory (DFT) and calculated cluster binding energies, showing positive ΔG values. The ability to control analyte CoVs by adjusting modifier concentrations in isocratic and gradient modes is beneficial for optimizing multidimensional LCxDMS–MS. It is fast and effective for manipulating the DMS scanning window size to realize shorter mass spectrometry (MS) acquisition cycle times while maintaining a sufficient number of CoV steps and without compromising DMS separation performance.