Evaluation of astrocyte proliferation in human brain pathology plays a key role in the diagnostic and prognostic assessment of diseases, especially in cases of benign or malignant brain neoplasms. However, the proliferative potential of nonneoplastic astrocytes within the affected human brain parenchyma has not been well defined. Given the beneficial functions of proliferating reactive astrocytes for brain repair in experimental models of stroke and trauma, investigating the context-specific potential of human astrocytes to proliferate will be a major step forward in exploiting their roles in the genesis, progression, and outcome of neurological diseases in patients. Here, we describe a step-by-step protocol for immunofluorescent staining and neurosphere-forming assay tailored to uncover the astrocyte proliferation and neural stem cell properties in samples of human brain tissue obtained during neurosurgical resections or in a small brain biopsy. This protocol allows for reliable assessment and evaluation of adoptive astrocyte plasticity in the context with various neuropathological conditions in the human brain.