Cholesterol homeostasis is highly regulated in the nervous system; dysregulation in cholesterol trafficking and content have been involved in the pathogenesis of neurodegenerative diseases (such as Parkinson's and Alzheimer's diseases). Furthermore, low cholesterol levels during brain development are associated with neurodevelopmental deficits and mental retardation. The methods described in this chapter can be used to investigate the effect of neurotoxicants on cholesterol homeostasis. Astrocytes and neurons are two major cell types in the brain in which cholesterol synthesis and efflux are highly regulated to keep a proper cellular cholesterol level. Disruption in cholesterol synthesis and/or cholesterol efflux may result in cholesterol deficiency or accumulation in these cells leading to brain dysfunctions.