The tegument (outer surface) of Schistosoma mansoni and other trematodes is in intimate contact with the host and plays an important role in host-parasite interactions. It is a complex structure that contains hundreds of proteins implicated in a variety of functions, although, so far, only a few proteins have been well characterized. Indeed, a few of these proteins have been shown to be effective vaccine and diagnostic candidates against S. mansoni and other schistosomes, and so the proteomic characterization of tegumental molecules could open new avenues for the development of novel control and surveillance strategies to combat schistosomiasis. Here, we describe the step by step isolation of tegumental proteins from the different tegument compartments using a biotinylation approach, as well as the materials and reagents needed.