New TLC, HPLC and LC/MS methods were developed for the rapid separation, characterization and quantitation of thiophenes in Radix Echinopsis, a herbal medicine, which has been used in China for long history. Nineteen commercial batches of this herb were analyzed by TLC and HPLC. Only five batches were derived from correct species. The other 14 batches were identified as its adulterating species, Radix Rhapontici, as they did not contain characteristic thiophenes of Echinopsis species. By LC/MS, 16 thiophenes were identified from the methanol extracts of Radix Echinopsis and Radix Rhapontici according to their fragmentation behaviors in MS/MS. In addition, a fully validated HPLC method was established to determine the contents of alpha-terthienyl in Radix Echinopsis. The contents ranged from 2.7 to 5.2 mg/g for five tested samples. The methods in this paper were simple and reliable, and could be used for the comprehensive quality control of Radix Echinopsis.