This paper presents the first direct delivery of silica beads into separation columns using bead/photoresist mixture for on-chip Liquid Chromatography (LC) – Electrospray Ionization (ESI) applications. Instead of packing beads into an existing column from off-chip, this new method uses photoresist mixed with surface-functionalized LC silica beads for a modified photolithography (including spin-coat, exposure and development) and parylene-channel process, followed by sacrificial photoresist removal (by acetone) to release the silica beads inside the LC column. The substantiation and validation of this new technique is demonstrated on an LC-ESI chip integrated with particle filters, silica-bead LC column, and electrospray nozzle, which has been tested both physically and chemically.