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Water-in-oil (w/o) emulsions have shown a promising release profile of small drug molecules and proteins. However, the major concerns are the structural stability, the retention of the activity and to avoid unwanted immunological reactions caused by the changes in protein structure. In the present study, the secondary structure of insulin and growth hormone is investigated after manufacture of w/o emulsions, using Fourier transform infrared (FTIR) spectroscopy. Initial investigations indicate an altered distribution in the secondary structure elements, e.g. α-helix and β-sheet, measured by area overlap calculations. The changes are more pronounced for growth hormone than for insulin. The overlapping area is 0.93±0.01 for the emulsion containing insulin manufactured at 0 °C and homogenised for 3 min, the corresponding value for growth hormone is 0.83±0.01. The droplet size changes from 0.27±0.04 μm in the blank w/o emulsion to 0.79±0.13 and 0.66±0.21 μm when insulin or growth hormone is incorporated into the w/o emulsions, respectively.