Gliomas, the heterogeneous primary brain tumors lacking efficient treatment, interact with their tumor microenvironment (TME) and the central nervous system (CNS) resident cells to support their own survival and growth. Deeper knowledge on these interactions is needed to improve treatment, and therefore glioma models incorporating components of human TME are of great interest. The aim of this work was to develop a three-dimensional (3D) in vitro coculture model for assessing physical and functional neuro-glioma interactions. For that, human glioblastoma cell line LN229/GFP-derived spheroids were cocultured with pre-established human induced pluripotent stem cell (hiPSC)-derived cortical neuronal network in collagen I hydrogel for 2-4 weeks. Cellular interactions were assessed by immunocytochemistry, calcium imaging and protein secretion analysis. Results revealed that the established model was viable, repeatable, and enabled formation of physical cellular connections. Calcium activity was observed in both cell types, indicating that the model environment supported typical cellular functionality. Also, protein secretion levels differed between mono- and cocultures, suggestive of paracrine signalling in cocultures. Next, a common chemotherapeutic agent temozolomide (TMZ) was applied. TMZ prevented glioma growth without disrupting neuronal networks or their functionality. As conclusion, this work demonstrates establishment of a feasible coculture model providing possibility to assess neuro-glioma interactions in 3D also on functional level and to study treatment response considering also the safety of non-malignant host cells.