Esterified cholesterol content is often lower than free cholesterol content in biological systems and thus the determination of the esterified cholesterol content of cells is often challenging. Traditional methods use enzymatic assays in which an indirect measurement of the esterified cholesterol content is obtained by subtracting the measurements of the free from the total cholesterol content. However, this approach fails in the case where the total cholesterol content of cells is unchanged while the ratio of free to esterified cholesterol changes such that total and free cholesterol content are very similar and thus the difference may fall within the background noise of the enzymatic assay. To overcome this challenge, we here describe a method that utilizes a TLC-based technique to isolate esterified cholesterol. Isolated esterified cholesterol can then be measured using traditional enzymatic methods. Therefore, this method provides a practical and more sensitive assay to measure esterified cholesterol content in cellular extracts.