Publisher Summary This chapter describes the assays to measure invertase enzymatic activity, focusing on the secretion of invertase fusion proteins as a reporter in studies of vacuolar protein trafficking. As a reporter protein, invertase enzymatic activity can be measured by both quantitative and qualitative assays. In addition, the invertase protein can be followed by methods that measure the extent and types of glycosylation it receives. Several important features of invertase have contributed to its utility in protein trafficking studies. Its enzymatic activity is not altered when fused to heterologous proteins by recombinant DNA techniques, and because invertase secretion is not essential under standard growth conditions, it can be easily manipulated without affecting cell viability. In addition, other than the N-terminal signal sequence that is cleaved after its translocation into the endoplasmic reticulum (ER), invertase does not appear to contain any sequence information that is essential for its transport through the secretory pathway. Thus, invertase enzymatic activity is accurately localized within the cellular compartment(s) to which the native protein under study is transported.