Immunostimulation represents a promising approach designed to specifically eradicate malignant cells. Since glioma tumour cells hole up in the central nervous system (CNS) in a particularly inauspicious milieu to antitumour immune reactions we here propose a new strategy to revert the properties of this microenvironment by administering an antitumour cytokine into the CNS tumour itself. Thus, biodegradable poly(d,l-lactide-co-glycolide) (PLGA) sustained-release microspheres for stereotaxic implantation loaded with interleukin-18 (IL-18), that is known to exert antitumour activity and trigger immune cell-mediated cytotoxicity, were developed. Different tests for assessing IL-18 bioactivity were set-up and evaluated. A specific bioassay was considered as the most reliable test. The stability and integrity of IL-18 was then verified during the encapsulation process. Consequently, two procedures of IL-18 encapsulation in PLGA microparticles (W/O/W and S/O/W) were investigated. As determined by radiolabelling studies using 125I-IL-18 and a continuous flow system, the in vitro release profile of IL-18 was optimum with S/O/W method with a moderate burst effect and a subsequent progressive discharge of 16.5 ± 8.4 ng/day during the next 21 days against 6.1 ± 4.2 ng/day with the W/O/W method. Considering analytical testing of IL-18 together with its preserved biological activity after release from microspheres, amounts of the active cytokine obtained with S/O/W method were relevant to plan in vivo evaluation to validate the therapeutic strategy.