The major aim of this study was to get deeper insight into the process of polymer cross-linking and the resulting structure of beads based on chitosan (CS) or chitosan/poly(ethylene oxide) (CS/PEO) semi-interpenetrating networks (semi-IPNs) as new carrier materials for oral drug delivery. Spherical hydrogels were prepared by a dropping method. The uptake kinetics of the cross-linking agent glyoxal into the beads were monitored and quantitatively described using Fick’s second law of diffusion. High-resolution synchrotron infrared microspectroscopy (SIRM) was used to characterize the inner structures of the beads. Importantly, the diffusion of glyoxal through the hydrogels was found to be much slower than the cross-linking reaction and the mesh size of the created networks to be much larger than the hydrodynamic diameter of glyoxal. The presence of PEO chains slightly decreased the diffusivity of glyoxal due to obstruction effects. However, the cross-linking reaction was not affected. Interestingly, the polymers were homogeneously cross-linked throughout the beads, except for a thin outer shell showing an elevated cross-linking density. Thus, the obtained cross-linked hydrogel-based beads exhibit well-defined polymeric structures and offer an interesting potential as novel oral drug delivery systems.