Abstract: This report is concerned with the practical application of thin-layer chromatography on alumina without binder for the separation and identification of steroidal sapogenins. After a series of experiments, the following preliminary conclusion was drawn. Neutral alumina of an activity Ⅲ or Ⅳ, with particle size smaller than 140 or 160 mesh was found to be the most suitable adsorbent. Mixtures of methanol and benzene were used as developing agents. Methanol and benzene with a ratio of 0.5 : 99.5 was adapted to separate monohydroxyl nonketonic steroidal sapogenins, while 2.5:97.5 was better for monohydroxyl nonketones and ketones and 5:95 for dihydroxyl nonketones and ketones. A 25% alcoholic solution of phospho-molybdic acid was a satisfactory spraying agent. Under the above condition, eight spots were shown by the crude mixture of steroidal sapogenins from Agave americana L.