Toad skin is a traditional Chinese medicine for the treatment of various tumors. The major active components in toad skin are bufadienolides. In this paper, a simple, accurate and reliable method for the simultaneous separation and determination of nine active components (gamabufotalin, arenobufagin, telocinobufagin, desacetylcinobufotalin, bufotalin, cinobufotalin, bufalin, cinobufagin and resibufogeni) in toad skin was developed using High-Performance Liquid Chromatography (HPLC) coupled with Photodiode Array Detection (PDA) detection. The chromatographic separation was performed on a SinoChrom ODS-BP C18 column with gradient elution using acetonitrile and 0.1% acetic acid-0.5% potassium dihydrogen phosphate aqueous solution at a flow rate of 0.8 mL min-1. All compounds showed good linearity in a wide concentration range with the values of r2 higher than 0.9994, and their limits of detection were at the range of 0.06-0.10 μg mL-1. The separation and identification of the nine bufadienolides in this paper may provide important experimental data for further research and application of toad skin.