Echinacea purpurea (L.) Moench, a top selling botanical medicine, is currently of considerable interest due to immunomodulatory, anti-inflammatory, antiviral and cannabinoid receptor 2 (CB2) binding activities of its alkylamide constituents. The purpose of these studies was to comprehensively profile the alkylamide (alkamide) content of E. purpurea root, and to compare yields of alkylamide constituents resulting from various ethanolic extraction procedures commonly employed by the dietary supplements industry. To accomplish this goal, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was validated for quantitative analysis of several E. purpurea alkylamides. Using this method, at least 15 alkylamides were identified and it was shown that fresh and dry E. purpurea extracts prepared from equivalent amounts (dry weight) of roots, with exceptions, exhibited similar yield of specific alkylamides. However, the amount of total dissolved solids in the dry extract was higher (by 38%) than the fresh extract. Two extracts prepared from dried roots at different ratios of root:solvent (1:5, w:v and 1:11, w:v) were similar in yield of total dissolved solids, but, there were differences in quantities of specific alkylamides extracted using these two root:solvent ratios. In addition, the important bioactive dodecatetraenoic acid isobutylamides are fully extracted from dry E. purpurea root in 2 days, suggesting that the manufacturing practice of macerating Echinacea extracts for weeks may be unnecessary for optimal alkylamide extraction. Finally, the identification of a new alkylamide has been proposed. These results demonstrate the differences of the described extractions and utility of the analytical methods used to determine the wide-ranging individual alkylamide content of commonly consumed Echinacea extracts.