A novel concept for assembling ordered porous silica microsphere monolayers for screening in miniaturized planar chromatography is disclosed. It contributes to sustainability in chromatography by facilitating parallel sample separation, reducing volume consumption, enabling cost-effective layer fabrication, and promoting the feasibility of automation. The first step toward realizing the proposed concept has been taken. Monodisperse porous silica microspheres with nominal diameters of 5 and 9 µm are assembled in a close-packed single-layer on a hydrophobic carrier surface to restrict cross-flow between adjacent tracks of separated samples. In particular, the assembly of close-packed chromatographic silica or C18-coated monolayers is demonstrated for the first time using a solvent-free rubbing technique on polydimethylsiloxane-coated glass slides. However, the coating is prone to swelling and influences the separation. It caused strong retention of lipophilic compounds and front-elution of water-soluble compounds, resulting in poor separation. Next steps will focus on assembling or transferring the close-packed monolayers onto a more inert glass coating or carrier and on functionalizing the silica microspheres to utilize the miniaturized chromatographic separation fully. Hyphenation with planar bioassays and high-resolution mass spectrometry for sensitive targeted detection of active compounds will follow.