Mesenchymal stem cells (MSCs) culture fluids are frozen and thawed for long-term storage when needed for medical purposes. However, stresses accompanied with freezing and thawing can disrupt the tertiary structures of proteins, which need to be in a certain state to show their therapeutic effect, by affecting their internal interactions. Supercritical drying of MSCs culture fluids using carbon dioxide was conducted to resolve these problems. Various process parameters were controlled to improve process efficiency in the water displacement step which takes place prior to supercritical drying. Among the proteins contained in the MSCs culture fluids, vascular endothelial growth factor (VEGF) was selected as a representative material to optimize the process parameters. Analyzed results show that the tertiary structures of VEGF via supercritical drying are well preserved. In addition, the structural stability and bioactivity of VEGF when re-dissolved in an aqueous environment were better than those obtained from freeze drying.