The protocol for preparation of methacryl-substituted polyhedral oligomeric silsesquioxane-based monolithic stationary phases containing zwitterionic sulfobetaine monomers in 450 μm i.d. fused silica capillaries is presented. The monolithic columns were evaluated by scanning electron microscopy, inverse size-exclusion chromatography, and isocratic HPLC separation of model compounds. Nature of zwitterionic sulfobetaine monomers has distinct impact on the structure of the final monolithic material as well as on its polarity and separation ability. The columns are suitable for multimodal chromatography as confirmed by their capability to separate compounds over a wide range of polarities (alkylbenzenes, phenolics, aromatic carboxylic acids, a mixture of purine-, pyrimidine-bases, nucleosides, and 2-deoxynucleosides). The critical mobile phase composition for the transition from hydrophilic interaction liquid chromatography to reversed-phase mode is around 84 % of acetonitrile which confirms the prevailing non-polar nature of formed monoliths. The proposed preparation protocol allowed preparation of stable columns whose efficiency reached the minimal height of the theoretical plate 6–10 μm which decreased by less than 12 % during the two months of everyday use.