Epidermal growth factor-like (EGF) repeats are found in numerous extracellular or transmembrane proteins including Notch. EGF repeats containing the appropriate consensus sequences can be modified with two unusual types of glycans: O-fucosylation and O-glucosylation. We have identified the glycosyltransferases that catalyze the addition of the first sugar to these consensus sites: protein O-fucosyltransferase 1 (Pofut1) and protein O-glucosyltransferase (Rumi/Poglut1). Recently, we have demonstrated that Rumi/Poglut1 shows protein O-xylosyltransferase activity as well. Here, we describe how we characterize the enzymatic activity of these enzymes, including preparation of the acceptor substrates, using bacterially expressed EGF repeats.