Bacterial glycosyltransferase toxins are secreted effectors which subvert normal cellular functions to enhance bacterial pathogenesis or survival. Recently a novel glycosyltransferase T3SS effector capable of transferring GlcNAc to Arginine residues was identified in enteropathogenic Escherichia coli (EPEC). NleB1 was shown to modify the death domain of FADD and TRADD and block extrinsic apoptosis signaling. To establish the true repertoire and kinetics of Arg-GlcNAcylation by NleB1, we developed an Arg-GlcNAcylation enrichment strategy. Utilizing a newly developed Arg-GlcNAc specific antibody we show endogenous targets of NleB1 can be effectively enriched at the glycopeptide level enabling both identification of glycosylation substrates and sites of modification. Using inducible stable cell lines, we found that multiple human proteins can be modified by NleB1 yet FADD appears to be the dominant and first Arg-GlcNAcylated target. Temporal profiling of Arg-GlcNAcylation showed that, during EPEC infection, alternative Arg-GlcNAcylation substrates only appeared after FADD modification or in response to overexpression of NleB1. Similarly, homologues of NleB1 such as SseK1 from Salmonella enterica serovar Typhimurium also appeared to target FADD at the conserved residue Arg117. However, within TRADD the death domain was modified at alternative sites by SseK1 compared to NleB1. Surprisingly, within both EPEC and S. Typhimurium, we noted the modification of multiple bacterial proteins including NleB1 and SseK1 themselves. These findings suggest FADD is the preferred target of NleB1 during EPEC infection and highlights the power of Arg-GlcNAc pulldowns for the identification and characterization of Arg-GlcNAcylating enzyme substrates.