Long-term stability is one of the main challenges for translation of therapeutic proteins into commercially viable biopharmaceutical products. During processing and storage, proteins are susceptible to denaturation. The aim of this work was to evaluate the stability of amphiphilic cyclodextrin-based nanoparticles (NPs) containing insulin glulisine. The stability of the NP dispersion was systematically evaluated following storage at three different temperatures (4¿°C, room temperature (RT) and 40¿°C). While the colloidal parameters of the NPs in terms of size and zeta potential were maintained (109¿±¿9¿nm, polydispersity index 0.272, negative zeta potential -25¿±¿3¿mV), insulin degraded over 60 days during storage. To enhance the shelf life of the product and to circumvent the need for cold-chain maintenance, a lyophilized formulation containing insulin glulisine NPs (1.75¿mg/mL of NPs) and 25¿mg/mL trehalose was produced. The freeze-dried powder extended the stability of the product for up to 30 days at ambient temperature and 90 days at 4¿°C (with 95% and >80% insulin recovery, respectively). Following intra-intestinal administration of the freeze-dried formulation, while no lowering of blood glucose was seen, insulin glulisine was detected in both portal and systemic blood indicating that potential exists for further development of the formulation to simultaneously achieve prolonged stability and therapeutic efficacy.