AbstractDrug-loaded proteinoid microspheres were freeze-dried to facilitate shipping and handling and to enable long term storage. Heparin was chosen as the model drug in developing the optimum lyophilization process. The factors influencing the integrity of either heparin-loaded or unloaded (‘empty’) proteinoid microspheres during freeze-drying were determined, with emphasis on: selecting an optimum freezing and resuspending temperature; choosing an appropriate cryoprotectant and its optimum concentration in the formulation; and, designing a suitable method for formulating the microspheres. Freezing at/below -70°C was found to minimize damage to the microspheres. Addition of sugars, such as trehalose and lactose, as cryoprotectants, further increased the stability of the heparin-loaded microspheres during freeze-drying. The optimum trehalose or lactose concentrations were determined to be 5% (w/v). Using the optimumized lyophilization process described in this manuscript, microspheres remained intact during freeze-drying. The freeze-dried microspheres were stable for at least three months post-lyophilization.