The blood brain barrier (BBB) consists mainly of endothelial cells, astrocytes and pericytes embedded in a specialized basement membrane. To properly evaluate the ability of novel brain tumour therapeutics to cross the BBB, an ‘all human’ model that better reflects in vivo conditions, critical for the BBB function and integrity has been developed to assess the delivery of modified Kytozyme nanoparticles (NPs). Human brain endothelial cells (hCMEC/D3), human cortical astrocytes (SC-1800) and human brain vascular pericytes (HBVP) in mono-, co- and tri- cultures were plated and TEER values were measured using an Electric Cell-Substrate Impedance Sensing (ECIS) system. Tight junction protein levels (ZO-1, occludin and β-catenin) were examined by Western blotting and immunocytochemistry. Response of culture combinations post NPs addition was assessed using cytotoxicity assay. Barrier integrity post-injection of NPs loaded in Evans Blue was monitored using live cell imaging. Co-cultures and tri-cultures resulted in significantly higher TEER values compared to endothelial cells alone (p < 0.05). Maximal TEER values for each condition were observed at 120 hours post plating. TEER values for co-culture (E + A) were 1250 Ω/cm2, and for tri-culture (E + A + P), 1190 Ω/cm2 compared to monoculture (E) (950 Ω/cm2). These peak values lasted for an additional 125 hours and provided plenty of time to test NP delivery. Western blot analysis revealed that ZO-1, occludin and β-catenin protein expression was higher in co- and tri-cultures compared to mono culture. In addition, ICC studies revealed localised expression of these tight junction proteins at cell-cell contacts whereas in monoculture, diffuse staining was observed. Following addition of Kytozyme NPs to the co-culture (E + A), at 120 hours, there was a decrease in TEER values (1250 Ω/cm2 to 1120 Ω/cm2) with a full recovery to peak TEER values within 12 hours suggesting passage through this BBB model. The addition of Evans Blue had no effect on TEER values. This study not only confirms that astrocytes contribute to BBB formation in terms of higher TEER values and in higher expression of ZO-1, occludin, and β-catenin proteins which are involved in tight junction formation but also provides an effective model with which to study nanoparticle drug delivery. We are now loading NPs with drugs to provide meaningful data that should help bridge the gap between drug discovery and pre-clinical studies.