The Calcium-dependent interaction between guanylyl cyclase-activating proteins (GCAPs) and retinal guanylyl cyclase (ROS (rod-outer-segment)-GC) is involved in processing light stimuli in the visual sensory cells of the retina. Malfunction of this interaction is associated with different kinds of retinopathies. So far, the interaction between both proteins is barely understood on a molecular level. The combination of chemical cross-linking and high-resolution mass spectrometry (MS) presents an alternative approach for the structural characterization of proteins and protein complexes. In this work, the interaction between binding motifs derived from ROS-GC and GCAP-2 as well as the homodimerization of GCAP-2 were investigated by different cross-linking strategies (amine-reactive, photo-reactive, MS/MS-cleavable). The results allowed creating the first structural models of GCAP-2/GC peptide complexes as well as the GCAP-2 homodimer.