Liposomes are widely used vehicles for the delivery of bioactive molecules. They are composed mainly from acyl-phosphatidylcholines, cholesterol, and charged lipids (e.g., stearylamine, dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine). The incorporation efficiencies of the bioactive molecule and the drug to lipid molar ratio are important factors for the assessment of the liposomal formulation. In order to successfully characterize a liposomal formulation, it is necessary to be able to accurately measure the lipids and the encapsulated molecule, using the smallest possible sample. The present work describes an analytical methodology on qualitative and quantitative determination of all the lipid ingredients that are involved in the liposome formulation, as well as the drug incorporation and the drug-lipid ratio, by a simultaneous measurement of all the liposomal ingredients using thin-layer chromatography coupled with a flame ionization detector (HPTLC/FID). The procedure requires only one measurement per sample, and it can be applied even in very small or much diluted samples. The proposed analytical method can be applied in general on all steps of the development of liposomal formulations. The purity and stability of the raw materials can also be easily evaluated. In addition the preparation procedure can be tracked in order to locate possible losses of raw material and errors of the preparation method resulting in the amelioration of the method.