Understanding the mechanism of developmental brain injury is crucial for the progress of discovering neuroprotective strategies and interventions. However, the pathophysiology is complex which involves interactions and crosstalk of diverse neural cell types. Isolating viable and pure populations of these brain cells is a valuable tool to study the particular cell properties and understand the physiologic and pathophysiologic mechanisms. Here we present a magnetic cell sorting approach to separate astrocytes and neurons sequentially from the same neonatal (postnatal day 9 or 10) CD-1 mouse brain samples. The procedure which involves positive selection of astrocytes by the ACSA-2 antibody followed by a negative depletion of non-neuronal cells from the flow through yields relatively enriched neuronal cells. The sorted fractions are highly pure and viable and can be used for further applications and analyses.