We present a sensitive high performance liquid chromatography (HPLC)-based method for the determination of free cholesterol in brain tissue and cell cultures. The method does not require the derivatization of the analyte and uses separation and quantification by reversed-phase HPLC coupled to UV detection. Lipids are methanol/chloroform extracted following the method of Bligh and Dyer, and separated using isopropanol/acetonitrile/water (60/30/10, v/v/v) as mobile phase. Lineal detection is observed in a wide range of concentrations, from 62.5 to 2000 ng/µL. The complete method was validated measuring a significant cholesterol increase in the brains of C57BL6 mice between postnatal days 2 and 10, and a significant cholesterol decrease in glial cells in culture treated with an inhibitor of the protein deacetylase SIRT-1. We consider this analytical method a useful tool to assess free cholesterol levels in brain samples and cell cultures.