Protein O-glycosylation is involved in numerous biological processes. Deconvoluting the functions of O-linked glycans requires having glycosyltransferace (Gtf) inhibitors suitable for use in cells. Although high-throughput screening has provided small-molecule inhibitors suitable for dissecting kinase pathways, it has not been applied to discover inhibitors of protein glycosylation due to a lack of suitable assays. Here we describe a general protease-protection assay in which a peptide labeled with a FRET pair is subjected to glycosylation and then treated with a protease that discriminated between glycosylated and non-glycosylated peptides. We show that this approach can be adapted to monitor the activity of O-GlcNAc transferase (OGT), a Gtf involved in intracellular signaling, as well as two O-GalNAc transferases (ppGalNAc T1 and T2), which initiate O-linked mucin-type glycosylation. We have used the strategy to screen 124 226 small molecules against OGT and have discovered several low micromolar inhibitors. It should be possible to use this assay to discover inhibitors for many Gtfs that catalyze O-linked glycosylation, which will enable researchers to assess the potential of non-substrate-based small molecules for dissecting glycosylation pathways in cells.