This report details the fabrication of polystyrene-based nano-LC monolithic columns for the separation of protein molecules. The report focuses on the practical advantages of monolithic columns when compared to conventional packed columns. Capillary columns were made to 100 and 50 microm i.d. and used to analyse a mixture of proteins, these separations were then compared with a conventional protein phase under the same conditions. A second functionalised monolithic polystyrene-based column was also manufactured and compared for the analysis of proteins, under the same analytical conditions, to the standard non-functionalised bare polystyrene monolith. Nano-LC polystyrene monolithic columns were found to be advantageous to conventional phases for the analysis of protein molecules, with a one-step fabrication process, faster analysis times, lower limits of detection hence higher sensitivity.