Chitosan (CS) and chondroitin sulfate (CHS) are natural polymers with demonstrated applicability in drug delivery, while nanoparticles are one of the most explored carriers for transmucosal delivery of biopharmaceuticals. In this work we have prepared CS/CHS nanoparticles and associated for the first time the therapeutic protein insulin. Fluorescein isothiocyanate bovine serum albumin (FITC-BSA) was also used to enable comparison of behaviors regarding differences in molecular weight (5.7 kDa versus 67 kDa). Nanoparticles of approximately 200 nm and positive zeta potential around +20 mV were obtained. These parameters remained stable for up to 1 month at 4 °C. Proteins were associated with efficiencies of more than 50%. The release of FITC-BSA in PBS pH 7.4 was more sustained (50% in 24 h) than that of insulin (85% in 24 h). The biocompatibility of nanoparticles was tested in Calu-3 and A549 cells by means of three different assays. The metabolic assay MTT, the determination of lactate dehydrogenase release, and the quantification of the inflammatory response generated by cell exposure to nanoparticles have indicated an absence of overt toxicity. Overall, the results suggest good indications on the application of CS/CHS nanoparticles in respiratory transmucosal protein delivery, but the set of assays should be widened to clarify obtained results.