Abstract Glioblastoma (GBM) is a malignant and aggressive brain tumor that is difficult to treat due to its heterogeneity. Traditional in vitro models (i.e. monolayers and tumorspheres) do not recapitulate this heterogeneity, leading to difficulty assessing new therapeutic strategies. Culturing GBM cells as organoids may provide a better method to retain the phenotype of the parent tumor. Here, we present a protocol for generating GBM organoids using NeuroCult™ NS-A Proliferation Medium, a medium commonly used for tumorsphere cultures. Two methods were assessed for the initial stage of formation, each suitable depending on throughput and cell source. In the first method, tumorspheres were dissociated and single cells were mixed with Growth Factor Reduced Matrigel® at a 1:4 ratio to form 15 μL droplets with 1000 cells per droplet. In the second method, 9000 cells from dissociated tumorspheres were seeded per well in a 96-well U-bottom plate and cultured for 5 days to form aggregates. On day 6, the aggregates were embedded in 15 μL Matrigel® droplets. In both methods, polymerized droplets were then cultured in suspension in stationary 6-well plates for 3 days, then maintained on an orbital shaker with full-medium changes every 2 - 3 days. Organoids were cryosectioned and processed for immunofluorescence at 5 - 6 weeks. In vivo, GBM tumors display an architecture containing regions of high and low nutrient and oxygen availability around a necrotic core. GBM organoids generated using this protocol exhibited distinct areas of proliferation near the organoid rim (Ki67+) and hypoxia (CA9+) near the core, mimicking this in vivo architecture (n = 3 GBM cell lines). Organoids grew to 2 - 4 mm in diameter and expressed glioma markers including TUJ1, Nestin, SOX2, OLIG2, and GFAP. These data show that NeuroCult™ NS-A Proliferation Medium can be used to adapt traditional GBM culture systems into organoids.