Electrospray has been recognized as an efficient technique for the fabrication of polymer micro and nanosystems and recently it was applied to lipids. The objective of this study was to assess the potential of electrospray for the encapsulation of insulin into lipid particles. Spherical particles of about 1 µm were obtained jetting a propanolic solution of palmitic or stearic acid and ethylcellulose or Pluronic F127 in a 10:1 or 20:1 (w/w) ratio under an electric field of 30 kV. Insulin was entrapped into the particles with high encapsulation efficiency by the formation of an ion-pair with sodium dodecyl sulphate. Far-ultraviolet circular dichroism spectroscopy indicated that electrospray did not modify the secondary structure of insulin. An in vitro prolonged release over 24 hours was observed after an initial burst effect. This study demonstrates that electrospray represents a viable new alternative for preparing in a single step peptide-protein loaded lipid based microspheres directly in powder form.