During chronic treatment with L-dopa (LD), Parkinsonian patients often experience uncontrolled motor complications due to fluctuations of the plasmatic levels of LD that result in pulsatile dopaminergic stimulation. To overcome these plasmatic fluctuations, a novel prodrug of LD, L-dopa-α-lipoic acid (LD-LA), has been proposed as a tool for achieving continuous dopaminergic stimulation. Due to slower susceptibility toward enzymatic conversion by LD-degrading enzymes (such as catechol-O-methyltransferase and monoamine oxidase), the plasma half-life of this prodrug is longer than that of LD. Moreover, the higher lipophilicity of LD-LA over LD promotes its delivery to the CNS, where the resulting levels of dopamine (DA) are kept high for a longer time than after equimolar administration of LD. To further reduce fluctuations in plasma levels of LD, LD-LA has been entrapped into biodegradable polymeric microspheres to be used as a depot system with the aim to prevent prodrug degradation and to obtain a sustained release of the intact compound. In the present work, a formulation of LD-LA loaded microspheres (characterized for drug loading, size, morphology, thermal properties, and in vitro prodrug release) has been administered subcutaneously to rats, and the resulting levels of LD and DA in plasma and striatal tissue, respectively, have been monitored. A good correlation between the in vitro release kinetics and the time range during which the formulation alters the LD/DA tissue levels in vivo was observed, suggesting that the polymeric microsphere matrix protects the loaded prodrug from chemical and enzymatic degradation and controls its release. Interestingly, LD-LA microspheres provided sustained levels of DA neurotransmitter in the striatum nucleus for up to 4 days after a single administration. In conclusion, a polymeric microsphere formulation of LD-LA is an attractive medicine for treating Parkinson's disease (PD) symptoms, avoiding motor complications.