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In this work, the recombinant spider silk protein eADF4(C16) was used to fabricate particles in the submicron range using a micromixing method. Furthermore, particles in the micrometer range were produced using an ultrasonic atomizer system. Both particle species were manufactured by an all-aqueous process. The submicroparticles were 332 nm in average diameter, whereas 6.70 μm was the median size of the microparticles. Both particle groups showed a spherical shape and exhibited high β-sheet content in secondary structure. Submicro- and microparticles were subsequently steam sterilized and investigated with respect to particle size, secondary structure and thermal stability. Sterilization temperature and time were increased to assess the thermal stability of eADF4(C16) particles. Actually, particles remained stable and their properties did not change even after autoclaving at 134 °C. Both, the untreated and the autoclaved submicroparticles showed no overt cytotoxicity on human dermal fibroblasts after incubation for 72 h. The eADF4(C16) particles were already loaded with proteins and small molecules in previous studies. With that, we can provide a highly promising parenteral drug delivery system based on a defined polypeptide carrier, manufactured with an all-aqueous process and being fully sterilizable.