The aim of this study was to investigate the performance of a newly devised high-performance thin-layer chromatography (HPTLC) method in quantifying common liposome membrane components, including the five phospholipids (PLs), phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, as well as cholesterol, cholesteryl hemisuccinate, and linoleic acid. Besides strictly keeping to a standardized procedure, three parameters were particularly critical for proper quantification. First, a relative humidity of higher than 60% caused migration distances to increase and reduced the resolution of the PLs on a silica-gel 60 HPTLC plate. Second, PLs underwent oxidative combustion during storage for 2 or 24 hours on an HPTLC plate, with peak losses of up to 25-44%. These losses could be prevented by storage under nitrogen and, to some extent, by the addition of the antioxidant, DL-alpha-tocopherol. Third, even with automated sample application, the accuracy and consistency of the application volume proved to be an important cause of error and needs routine verification. Considering these parameters, the method was found to accurately and precisely determine the composition of three different liposome preparations. The recovery was 97.2-101.8%, compared to secondary methods, and consistent over different days and with different operators (mean RSD of the recovery: 2.03 +/- 1.16%, n = 9). The working range was determined to be 100-300 ng in the case of the PLs (individual limit of determination between 40 and 80 ng) and 20-60 ng in the case of cholesterol (limit of determination: 16 ng).