Basic proteins, an important class of proteins in intracellular organisms such as Leishmania, are usually underrepresented on 2D gels. This chapter describes a method combining basic proteins fractionation using Free flow electrophoresis in isoelectric focusing mode (IEF-FFE) followed by protein separation using two-dimensional gel electrophoresis (2-DE) in basic conditions. The combination of these two techniques represents a great improvement for the visualization of Leishmania proteins with basic pI using 2D gels.