An easy and reliable HPLC method was developed to determine allantoin in Dioscorea Rhizoma using cyano columns. Qualitative and quantitative analyses of allantoin were performed successfully by cyano columns (YMC-Pack CN column, Zorbax SB-CN column and Discovery$^{(R)}$ Cyano column). The intraday precision were 0.58 - 3.33% for YMC-Pack CN, 0.41 - 2.20 for Zorbax SB-CN and 0.45 - 1.93% Discovery$^{(R)}$ Cyano columns, while interday variations were 0.09 - 1.84%, 0.04 - 2.59% and 0.87 - 5.18% for YMC-Pack CN, Zorbax SB-CN and Discovery$^{(R)}$ Cyano columns. The recoveries of allantoin were in the range at 98.8 - 102.6% (RSD 1.1 - 1.6%) for YMC-Pack CN column, 99.7 - 110.5% (RSD 1.3 - 4.9%) for Zorbax SB-CN column, and 97.2 - 110.1% (RSD 1.8 - 5.7%) for Discovery$^{(R)}$ Cyano column. The contents of allantoin in four Dioscorea Rhizoma samples were determined by cyano columms and ranged at 4.1-7.1 mg/g dry weight. The present study indicated that HPLC method using cyano column for determining allantoin is a reliable method and this method can be applied to verify allantoin in Dioscorea Rhizoma.