Two-dimensional gel electrophoresis (2-DE) is the most popular and versatile method of protein separation among a rapidly growing array of proteomic technologies. Based on two independent biochemical characteristics of proteins, it combines isoelectric focusing, which separates proteins according to their isoelectric point (pI), and SDS-PAGE, which separates them further according to their molecular mass. An evolution of conventional 2-DE is represented by the 2D-Difference in Gel Electrophoresis (2D-DIGE) that allows sample multiplexing and achieving more accurate and sensitive quantitative proteomic determinations. The 2-DE separation permits the generation of protein maps of different cells or tissues and the study, by differential proteomics, of protein expression changes associated to the different states of a biological system. In order to identify the molecular bases of pathological processes, it is also useful to characterize the physiological protein homeostasis in healthy cells or tissues. On these grounds, the availability of detailed 2D reference maps could be very useful for proteomic studies. The protocol described in this chapter is based on the 2D-DIGE technology and has been applied to obtain the first 2-DE reference map of the human small intestine.