Supercritical Emulsion Extraction in a Continuous operation layout is proposed for the production of poly-lactic-co-glycolic acid (PLGA) microspheres loaded with insulin, selected as a model of bioactive signal. Microspheres with different mean sizes of 2 μm (±0.9 μm) and 3 μm (±2.2 μm) and insulin loadings of 3 and 6 mg/g were obtained by processing different water-oil-water emulsions; an encapsulation efficiency of about 60% w/w was measured in all cases. Insulin release profiles from PLGA microspheres were also characterized in two different media (Phosphate-Buffered Saline and Dulbecco's Modified Eagle Medium) and kinetic constants were estimated by using a model proposed in literature. The produced microspheres were, then, used for the cultivation of rat embryonic ventricular myoblasts in a serum-free medium to monitor the biological effect of the released insulin. The best cell viability and proliferation, supported by released insulin, was monitored when microspheres with mean size of 3 μm loaded with 3 mg/g of insulin were added.