Microporous organic networks (MONs) that exhibit good stability and hydrophobicity are promising candidates for performing HPLC separation of small organic compounds. However, their applications in separating large analytes as well as biomolecules are still limited by the microporous nature of MONs. Herein, we demonstrated the fabrication of a MON-functionalized silica (MON@SiO 2 ), exhibiting micro and mesopores for the HPLC separations of small drugs as well as large analytes, such as flavones, nonsteroidal anti-inflammatory drugs (NSAIDs), endocrine disrupting chemicals (EDCs), and proteins. MON was successfully modified on SiO 2 microspheres to yield the uniform and mono-dispersed MON@SiO 2 . The separation mechanisms and performance of the MON@SiO 2 packed column were evaluated for a wide range of analytes, including neutral, acidic, basic compounds, drugs, and proteins. Compared with commercial C18 and SiO 2 -NH 2 packed columns, the proposed MON@SiO 2 column afforded superior performance in the separations of flavones, NSAIDs, EDCs, and proteins. Moreover, the MON@SiO 2 column also offered good repeatability with intraday RSDs (n = 7) of <0.1%, <2.0%, <2.3%, and <0.7% for the retention time, peak height, peak area, and half peak width, respectively, for separating EDCs. This work proved the potential of using MONs in the HPLC separations of drugs and proteins.