Understanding the mechanisms of protein sorting and targeting through the plant secretory pathway has become the focus of many research laboratories. The development of a model system whereby recombinant genes can be transiently expressed in protoplasts has facilitated the study of protein transport signals. Experimental strategies combining a protoplast expression system with endoglycosidase H, vacuole purification, and pulse-chase analyses are used to investigate aspects of specific proteins as they pass through the secretory system. This chapter provides details of protoplast preparation and electroporation as well as techniques to study protein trafficking from the endoplasmic reticulum to the Golgi apparatus or vacuolar compartments. Recommendations as to how to troubleshoot problems that can arise while following these protocols are also discussed in this chapter.