Glioblastoma, a WHO Grade IV brain cancer, and neuroblastoma, the most common extracranial solid tumour in children, are tumours often diagnosed at an aggressive stage which can have poor long-term outcomes for patients (1). Nanomedicine (the medical application of nanotechnology) has shown potential to improve cancer therapies, using biocompatible drug delivery vehicles which can encapsulate chemotherapeutics and deliver them to the tumour site(s) (2). However, preclinical research in nanomedicine using two-dimensional (2D) cell models has been found to have low predictive power for evaluating nanoparticle designs, and are poorly representative of solid tumours that grow in a heterogeneous three-dimensional (3D) environment. Conversely, 3D tumour spheroids have been shown to emulate some key elements of the tumour environment, and thus present a better model for investigating nanoparticle penetration and delivery into tumours (3). The aim of this study was to establish fluorescent-labelled spheroid models of glioblastoma and neuroblastoma in order to develop a quantitative platform for measuring penetration kinetics in 3D tumour spheroids. Cy5-labelled porous silica nanoparticles (~30 nm) were used as a model compound in this analysis. Tumour cell spheroids were grown to 500 um in diameter and tracking live uptake of nanoparticles over time revealed accumulation of silica nanoparticles deep into these tumour spheroids within 12 hours using confocal microscopy. This was then analysed and quantified using radial averaging and diffusion kinetics. Results demonstrate different diffusion patterns depending on the tumour model and the coating used on the surface of the silica nanoparticle. Overall, this presents an analysis platform for quantifying visual data of nanoparticle uptake in 3D tumour models, with the capacity to evaluate and improve drug-loaded nanoparticle delivery for these two aggressive cancers in the future.