Effector glycosyltransferases are a growing class of secreted bacterial proteins which subvert normal cellular functions by the modification of host proteins with carbohydrates. Recently a novel family of T3SS glycosyltransferases were identified within the enteric pathogens enteropathogenic Escherichia coli (EPEC), Salmonella enterica serovar Typhimurium and Citrobacter rodentium which mediates the addition of N-acetylglucosamine to arginine residues. The archetype of this family, NleB1 of EPEC, has been shown to modify a range of death-domain-containing proteins including FADD, TRADD and RIPK1. Yet, these targets were identified under ectopic expression with the true targets during infection still unclear. To define the substrates Arginine-glycosylation during in vitro we have developed a Arg-GlcNAcylation affinity proteomic approach. Utilizing this approach, we have characterized the repertoire of Arginine-GlcNAcylation during NleB1 ectopic expression, EPEC infections under different level of NleB1 expression and C. rodentium infections. We show that multiple host targets can be Arginine-GlcNAcylated when non-authentic level of NleB1 are introduced, yet during EPEC and C. rodentium infections Arginine-117 of FADD is rapidly and preferentially subjected to modification. Temporal profiling of Arg-GlcNAcylation showed that during EPEC infections 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 demonstrate a restricted repertoire during infection compared to overexpression. These findings demonstrate that the NleB/SSeK effectors are far more promiscuous than previously thought and highlights the power of glycopeptide affinity enrichment to identify and monitor Arg-GlcNAcylated substrates.