PLGA microspheres with high drug loading and high encapsulation efficiency were fabricated by a novel solvent evaporation process-in-situ S/O/W process. Insulin was dissolved in DMSO and dispersed into DCM to form fine particles due to an anti-solvent effect. The in-situ formed suspension was then added into an aqueous phase and emulsified. Microspheres were formed following the evaporation of organic solvents. The experimental results showed that the modified S/O/W process could encapsulate more than 90%(w/w) insulin in the microspheres with a drug loading of over 15% and the initial burst was much less than microspheres made by a W1/O/W2 process. Compared with a traditional water-in-oil-in-water (W1/O/W2) process, the in-situ S/O/W process does not require high solubility of the encapsulated drug in water and, because no special pre-treatment is needed to reduce the particle size of the drug, it is superior to an ordinary S/O/W process. The in-situ S/O/W process is particularly applicable to encapsulate peptides and low molecular weight proteins.