This chapter focuses on post-translational modifications of secretory proteins. Proteins that traverse the secretory pathway and reach the plasma membrane may be targeted to various locations. One final destination is the plasma membrane itself. The plasma membrane contains not only many transmembrane proteins, but also a special group of proteins, which are C-terminally linked to the outer leaflet of the plasma membrane through a so-called “glycosylphosphatidylinositol” (GPI) anchor. Between the plasma membrane and the cell wall, periplasmic proteins, such as invertase and acid phosphatase, may accumulate. A limited number of proteins, such as chitinase, and several heat shock proteins are secreted into the medium. Secretory proteins are modified in various ways during their passage through the secretory pathway. The chapter discusses techniques to identify and characterize the post-translational modifications of secretory proteins. It is important to realize that these techniques, although primarily developed for Succhuromyces cereuisiae, are in many cases also valid for other Ascomycetes, including the filamentous fungi belonging to this taxonomic group. The chapter discusses the glycosylation of secretory proteins and the detection of N- and O-glycosylation in secretory proteins.