Neuropeptide Y (NPY) receptors comprise a family of rhodopsin-like G-protein coupled receptors (GPCRs) that participate in controlling food intake, memory retention, and circadian rhythm, making them highly attractive drug targets. However, the multiligand nature of NPY receptors, such as the NPY receptor type 2 (Y2R), requires a detailed understanding of the receptor’s interactions with their natural ligands. Binding assays have so far provided insights into multiple Y2R-NPY conformers with distinct binding affinities that might also be controlled by intracellular Y2R-Gi protein-protein interactions. However, structural biology approaches, such as X-ray crystallography or cryo-electron microscopy, have so far not been able to capture these Y2R-NPY conformers, nor have they been able to resolve the conformational states of Y2R’s N-terminus. The N-terminus of Y2R has been classified as intrinsically disordered region. Cross-linking mass spectrometry (XL-MS) was employed to characterize the different conformational states and binding modes of Y2R upon binding of its ligand NPY. Cross-linked peptide digests were analyzed on a timsTOF Pro instrument using Parallel Signal Accumulation and Fragmentation operated in data dependent acquisition (DDA) and data independent acquisition (DIA) modes. The combined effects of precursor ion accumulation in the TIMS cell, fragment ion current accumulation through fragmentation events stacking, and the additional ion mobility dimension of peptide separation by DDA-PASEF provided better fragment ion spectral evidence of low-intensity cross-linked peptides. At the same time, spectra generated by co-isolation of high-abundant unmodified peptides were minimized. The comprehensive evaluation of each cross-linked peptide’s chromatographic and ion mobility properties in Skyline, in addition to fragment ion spectra, greatly improved the confidence of filtered peptide identifications. For the Y2R-NPY interaction, multiple cross-linking sites were identified between Y2R and NPY, involving the disordered N-terminal region of Y2R. The coexistence of different cross-linking sites between Y2R’s N-terminus and different amino acids in NPY point to multiple conformational states of Y2R’s N-terminus. Our results provide first insights into the interactions of NPY with Y2R at a molecular level.