In this investigation, a method is developed for the isolation and purification of bufadienolides (resibufogenin, cinobufagin, and bufalin) from the Chinese traditional medicine ChanSu. The crude ChanSu extract is prepared with solvent refluxing under optimal conditions. A new chromatographic approach for separating bufadienolides utilizes silica gel column chromatography with isocratic elution using cyclohexane-acetone (5:1) as the mobile phase to separate resibufogenin, followed by reversed-phase C18 preparative HPLC column using isocratic elution of methanol-water (72:28) to separate bufalin and cinobufagin. Combining these two methods, bufalin and cinobufagin could be completely separated in high purity and recovery, and the amounts obtained were 1.9 and 3.1 g, respectively, from 500 g of ChanSu. These two compounds have been identified by mass spectrometry and 1H NMR, and their purities were quantitated by HPLC at 99% and 98%, respectively.