Lipids such as cholesterol, triacylglycerols, and fatty acids play important roles in the regulation of cellular metabolism and cellular signaling pathways and, as a consequence, in the development of various diseases. It is therefore important to understand how their metabolism is regulated to better define the components involved in the development of various human diseases. In the present work, we describe the development and validation of a high-performance thin layer chromatography (HPTLC) method allowing the separation and quantification of free cholesterol, cholesteryl esters, nonesterified fatty acids, and triacylglycerols. This method will be of interest as the quantification of these lipids in one single assay is difficult to perform.