Cationic polymers have been receiving much attention as non-viral gene vectors. The aqueous mobile phase was optimized in combination with Shodex OHpak SB columns for size-exclusion chromatography (SEC) analysis of disulfide-containing poly(ethylene imine) (PEI) derivatives used in gene delivery. Addition of acetonitrile in mobile phase was shown to be able to suppress the hydrophobic interactions between polymer analytes and the stationary phase. The absolute molecular weights and distributions of the cationic polymers were determined directly from online SEC-MALS (multi-angle light scattering)/RI (refractive index detector). The results demonstrate that a good SEC separation of disulfide-containing PEI derivatives used in gene delivery with little band broadening was achieved.