Subcellular fractionation is a valuable tool to follow protein traffic between cellular compartments. Here we detail a procedure for fractionating erythrocytes infected with the human malaria parasite P. falciparum using the bacterial pore-forming protein Streptolysin O (SLO). Additionally we describe an experimental protocol to determine protein topology by carrying out a protease protection assay on SLO-lysed infected erythrocytes.