We have examined the column chromatography using hydrophobic gel carriers, Sephadex LH-20 and HP-Cellulofine, for separation of ten toad poison bufadienolides: resibufogenin, cinobufagin, bufalin, bufotalin, cinobufotalin, telocinobufagin, desacetyl-cinobufagin, desacetyl-bufotalin, desacetyl-cinobu-fotalin, and gamabufotalin (Figure 1). As a result, the use of developing solvents such as n-hexane/CH2Cl2/MeOH(4:5:1), n-hexane/CHCl3/MeOH(4:5:1), and n-hexane/toluene/ MeOH (3:2:1) provided the effective separation of bufadienolides. For separation, we have established the flow speed of developing solvents and the relationship of separation with the diameter and length of column, in addition to the comparison of separation by Sephadex LH-20 and HP-Cellulofine and the relationship with silica gel thin-layer chromatography. Also, we have tested the separability of hydrophobic gel by repeated use and the recovery of bufadienolides on hydrophobic gel column by repeated use.