O -GlcNAc transferase (OGT) is the only enzyme that catalyzes the post-translational modification of proteins at Ser/Thr with a single β- N -acetylglucosamine ( O -GlcNAcylation). Its activity has been associated with chronic diseases such as cancer, diabetes and neurodegenerative disease. While numerous OGT substrates have been identified, its accepted substrate scope can still be refined. We report here an attempt to better define the peptide recognition requirements of the OGT active site using mRNA display, taking advantage of its extremely high throughput to assess substrate potential of a library of all possible 9-mer peptides. An antibody-based selection process is described here that is able to enrich an OGT substrate peptide from such a library, but with poor absolute recovery. Following four rounds of selection for O -GlcNAcylated peptides, sequencing revealed fourteen peptides containing Ser/Thr but these were shown by luminescence-coupled assays and peptide microarray to not be OGT substrates. By contrast, subsequent testing of an N-terminal tag approach showed exemplary recovery. Our approach demonstrates the power of genetic encoded libraries for selection of peptide substrates, even from very low initial starting abundance and under sub-optimal conditions, and emphasizes the need to consider binding biases of antibodies and both C – and N -terminal tags in profiling peptide substrates by high-throughput display.© 2020 Wiley-VCH GmbH.