Bovine serum albumin (BSA) was encapsulated into poly(lactide-co-glycolide) (PLG) microspheres by a solid-in-oil-in-water (s/o/w) technique. We tested whether perturbations in BSA secondary structure could be minimized during encapsulation by using trehalose and how this would influence BSA aggregation and release. BSA secondary structure was monitored noninvasively by Fourier-transform infrared spectroscopy. When BSA was co-lyophilized with trehalose, lyophilization-induced structural perturbations were significantly reduced. The formulation obtained (BSA-Tre) was encapsulated into PLG microspheres and, by optimizing critical encapsulation parameters, a loading efficiency of 85% was achieved. However, due to the loss of the excipient in the o/w emulsion step, the structure of BSA-Tre was more perturbed than before encapsulation. Excipient-loss and encapsulation-induced structural perturbations could be prevented by saturating the aqueous phase in the o/w step with trehalose and by using the organic solvent chloroform. This in turn reduced the formation of soluble BSA aggregates. BSA was released from PLG microspheres using the improved formulations with an initial release in 24 h of not more than 22%, followed by a sustained release over at least 2 weeks. In summary, optimization of the encapsulation conditions in the s/o/w procedure resulted in the encapsulation of BSA without procedure-induced structural perturbations and minimized the release of aggregated protein. This demonstrates that the s/o/w technique is an excellent alternative to the most common encapsulation procedure, namely the water-in-oil-in-water technique.