Abstract: The present paper reports investigations on thin layer chromatography for the identification of cardiac glycosides, using active alumina without binder. Factors influencing R_f values and shape of spots of cardiac glycosides have been studied. Satisfactory results were obtained with following appropriate factors: 1. Adsorbent-Neutral active alumina (Ⅲ) with particle sizes of 160-200 mesh. 2. Thickness of layer-1 mm. 3. Solvent system-Mixture of chloroform-methanol (99:1); (95:5); xylene-methyl ethyl ketone (1:1); Chloroform-dioxane-n-butanol (70:20:5) and ether. 4. Angle of inclination by the plate: 5-10°. 5. Distance between samples-1cm. 6. Spray reagent-Kedde and Raymond reagent. According to the results so far obtained, it is considered that thin layer chromatography can be used as an effective and rapid method for cardiac glycosides, especially monoglycosides and aglucones.