Two-dimensional gel electrophoresis (2-DE) is a protein separation technique often used to separate plasma or serum proteins in an attempt to identify novel biomarkers. This protocol describes how to run 2-DE gels using narrow pH 3-5.6 immobilised pH gradient strips to separate 2 mg of serum proteins. pH 3-6 ampholytes are used to enhance the solubility of proteins in this pH range before the serum proteins are separated in the first dimension by isoelectric point (isoelectric focusing) followed by molecular weight (SDS-PAGE). This approach using the pH 3-5.6 range differs from pH ranges more commonly used for serum or plasma biomarker discovery which span three or more pH units (e.g. pH 3-10 and 4-7), and has the advantage that the pH range lies outside the range of three highly abundant proteins and therefore improves separation and representation of low abundance features. The protocol described takes approximately 8 days.