Poly(styrene co-divinylbenzene) (PS-DVB) monolithic pre-columns of 50 μm inner diameter (ID) were developed for peptides and small molecules enrichment intended for use in automated miniaturized liquid chromatography-mass spectrometry (LC-MS) column switching system as alternative to 50 μm ID poly(butyl methacrylate-co-ethylene dimethacrylate) (BMA-EDMA) monoliths. Monomer/porogen ratio, percentage of good solvent, polymerization temperature, and polymerization time, and thermal initiator, were investigated in order to optimise the monolithic structure with a high surface area and good permeability. The efficiency was measured on 10 cm long column using a simple liquid chromatography ultraviolet (LC-UV) test system with toluene as the test analyte. In general, increasing polymerization temperature lead to a monolith with a higher number of small pores and backpressure. A ratio of 40/60 between monomers and porogens was required for a full structure of monolith. The columns made with LP yielded a better efficiency compare to the commonly used 2,2'-azobis(2-methylpropionitrile (AIBN) for both PS-DVB and BMA-EDMA monoliths. Reaction time strongly affected column efficiency. The best monolithic PS-DVB pre-columns were prepared, using a binary porogenic solvent of toluene (9%) and 1-decanol (51%), lauryl peroxide (LP) as initiator and polymerization temperature of 73°C for 2 hours (plate height, H = 90 μm). PS-DVB monoliths which provided good efficiency for toluene with reasonably backpressure gave a narrow elution peak for luteinizing hormone releasing hormone (LHRH) without breakthrough using gradient elution (10 cm length). The developed PS-DVB monolith gave better peak shape, trapping ability and loadability for peptides than a BMA-EDMA monolith using the solid phase extraction tandem mass spectrometry (SPE-MS/MS) system. When combining a PS-DVB monolithic pre-column (50 μm × 4 cm, 500 nl/min flow rate) with a porous layer open tubular (PLOT) PS-DVB analytical column (~0.75 μm film thickness, 10 μm × ~5 m, 40 nl/min flow rate), a longer retention time (tR) (~48 min) than expected was obtained. Thus, further development of a suitable pre-column for this system is needed.