<p>Microporous organic network (MON) has emerged as efficient stationary phases for high-performance liquid chromatographic (HPLC) separation. Nevertheless, the inherent superhydrophobic networks made their applications limited in the reversed-phase (RP) mode. In this work, a novel amino acid L-cysteine (L-Cys) modified core-shelled microsphere MON@SiO₂-L-Cys was synthesized via the efficient thiol-yne “click” reaction for hydrophilic interaction chromatography /reversed-phase liquid chromatography (HILIC/RPLC) mixed-mode chromatographic separation. Baseline separation of various hydrophilic and hydrophobic compounds including aromatic ketones, phthalate esters, alkylbenzenes, amines, polycyclic aromatic hydrocarbons, phenols, nucleosides, nucleic acid bases, sulfonamides, water-soluble vitamins, cephalosporin antibiotics, and estrogens was realized on MON@SiO₂-L-Cys packed column based on the predesigned mixed-mode retention mechanisms. The MON@SiO₂-L-Cys packed column also exhibited higher selectivity compared to bare SiO₂, MON@SiO₂ without L-Cys, and C₁₈ packed columns, revealing the synergistic interactions of MON’s networks and the modified L-Cys molecules. This work provided a feasible way to construct a novel amphipathic MON-based stationary phase, broadened the application scope of MON for hydrophilic analytes, and uncovered the prospects of MON in mixed-mode HPLC.</p>