Two-dimensional electrophoresis (2-DE) is a high-resolution technique for analysis and comparison of complex protein mixtures. With the advent of recent technical developments, its application has become significant in a wide range of fields. This chapter describes a proteomic approach for the analysis of metastasis-associated proteins using pre-fractionation of glycosylated proteins via lectin (HPA) affinity chromatography prior to separation by 2-DE. Guidelines for the preparation and storage of buffers, experimental conditions and protocols of affinity chromatography, isoelectric focussing, and SDS-PAGE conditions are provided. Critical parameters associated with the different steps of 2-DE are discussed.