Objectives: To determine whether the size of the spheres and the availability of nutrients affect the energy status of spheroids formed from D54 glioblastoma cells. Study design: In vitro study- controlled experiment. Material and Methods: Spheres of te commercially available glioblastoma cell line D54 were cultured. Spheres were cultured for 8 and 13 days to ensure growth and sphere formation. One set of spheres had its medium changed regularly, while the other set of spheres was grown without changing the medium. The evaluation of the energy status of the spheres grown under different conditions was determined using the colorimetric ATP test, the percentage of LDH released in the medium and the measurement of the intensity of the TMRE color depending on the mitochondrial activity. Data analysis was done by one-way ANOVA analysis using the MedCalc program. Results: Colorimetric analysis determined the highest concentration of ATP in the spheres after 8 days of cultivation with constant medium change compared to other sets of spheres. LDH analysis indicates impaired integrity of the cell membrane in the spheres that were grown without changing the medium, which increases with the increase of the spheres through extended cultivation time. Spheres with regular medium changes lead to impaired cellular integrity in some of the cells, which increases after 13 days. The highest mitochondrial activity is visible in the spheres that were grown with a constant source of nutrients, in contrast to the spheres that were exposed to the nutrient reduction, where drop in fluorescence was noticed, i.e. reduced mitochondrical activity. Conclusion: The highest energy status was recorded in D54 spheres after 8 days of growing, with regular media changes compared to the other set of spheres. The availability of nutrients from the medium proved to have the greatest impact on the decline in energy status.