© 2020 Elsevier B.V. Development of spray drying of biomolecules has proved challenging primarily due to protein instability caused by high drying temperatures and atomization process. This study suggests a two-step approach towards a better understanding of spray drying process of large biomolecules. Using lysozyme as a model biomolecule a full factorial Design of Experiments (DoE) was employed to define the most critical process and formulation parameters. With respect to parameters such as feed rate, outlet temperature (Tout) and feed solid concentration, Tout was determined to be the most significant factor affecting the enzymatic activity. Forced deactivation studies were performed to identify critical points of potential enzymatic degradation during the drying process. Results show that lysozyme in solid form significantly inactivated when heated up to temperatures similar to Tout (70¿90 °C). The approaches presented herein provide an improved understanding of the critical process and formulation parameters required for the successful spray drying of biomolecules.