<jats:p>Alkamides are suspected to contribute to the activity of Echinacea preparations. They are mainly derived from undeca‐ and dodecanoic acid and differ in the degree of unsaturation and the configuration of the double bonds. In total, 6 alkamides have been isolated from the roots of Echinacea angustifolia as major lipophilic constituents and have been investigated regarding their pharmacokinetics. A sensitive and specific method has been developed for the identification and quantification of these alkamides in human plasma using liquid chromatography electrospray ionization ion‐trap mass spectrometry. The method was applied to analyze plasma samples obtained from a randomized, open, single‐dose, crossover study after oral administration of a 60% ethanolic extract from the roots of E. angustifolia to 11 healthy subjects. The maximum concentration of dodeca‐2E,4E,8Z,10E/Z‐tetraenoic acid isobutylamides, the main alkamides in the roots of E. angustifolia, appeared already after 30 minutes and was 10.88 ng/mL for the 2.5‐mL dose.