Background: The neurosphere assay is a powerful in vitro tool to investigate neural stem cells in the dorsal lateral ventricle (dLGE). In the dLGE, metrics of sizes and numbers of neurospheres generated using this assay has not been completely characterized. The objective of this protocol is to provide a stepwise method from a single isolation that predicts the average number of neurospheres generated and to estimate an approximation of its sizes after several days in vitro. The advantage of this protocol is that no expensive and specialized equipment is needed for tissue isolation. Estimates about the numbers and sizes of neurospheres will provide investigators with quantitative data to advise on how much starting dLGE tissue is required to generate the appropriate number of spheres for the implementation of downstream applications, including immunocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro (DIV). Furthermore, we observed that this method yields an average range of neurospheres sizes greater than 50 μm, but less than 100 μm after 7 DIV. Lastly, using an anti-GFAP antibody, we show that these neurospheres can be stained, confirming their use in future immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of primary neurospheres that will be useful for further advances in the area of stem cell biology.