In this work, we aimed to characterize the surface and the internal structure of mannitol microspheres containing chitosan/tripolyphosphate nanoparticles, which were prepared by spray-drying. These microspheres were recently proposed as valuable candidates to transport therapeutic protein-loaded nanoparticles to the lungs owing to their favorable aerodynamic properties. To observe the distribution of chitosan nanoparticles and mannitol in the microspheres, specific characterization techniques, such as confocal laser scanning microscopy, X-ray photoelectron spectroscopy, and time-of-flight secondary ion mass spectrometry, were used. Results showed that mannitol is distributed in the whole particle and nanoparticles are homogeneously mixed with mannitol. Moreover, both components were detected in the microsphere surface, mannitol being present to a higher extent, which is in agreement with the theoretical mannitol/nanoparticle ratio of microspheres (80/20). Therefore, this work confirmed that chitosan nanoparticles were successfully encapsulated in mannitol microspheres, providing a homogeneous distribution of the nanoparticles and, hence, of the nanoencapsulated therapeutic macromolecule.