Roundabout 1, or Robo1, is a cell surface signaling molecule important in axon guidance. Its interaction with heparan sulfate (HS) and members of the Slit protein family is essential to its activity, making characterization of these interactions by structural methods, such as NMR, highly desirable. However, the fact that Robo1 is a glycosylated protein prevents employment of commonly used bacterial hosts for expression of properly glycosylated forms with the uniform 15N, 13C, and 2H labeling needed for NMR studies. Here, we apply an alternative methodology, based on labeling with a single amino acid type and high structural content NMR data, to characterize a two-domain construct of glycosylated Robo1 (Robo1-Ig1-2) interacting with a synthetic HS tetramer (IdoA-GlcNS6S-IdoA2S-GlcNS6S-(CH2)5NH2). Significant chemical shift perturbations of the crosspeak from K81 on titration with the tetramer provide initial evidence for the location of a binding site and allow determination of a 255 μM disassociation constant. The binding epitopes, bound conformation, and binding site placement of the HS tetramer have been further characterized by saturation transfer difference (STD), transferred nuclear Overhauser effect (trNOE), and paramagnetic perturbation experiments. A model of the complex has been generated using constraints derived from the various NMR experiments. Postprocessing energetic analysis of this model provides a rationale for the role each glycan residue plays in the binding event, and examination of the binding site in the context of a previous Robo-Slit structure provides a rationale for modulation of Robo-Slit interactions by HS.