BACKGROUND The medicinal plant Spilanthes acmella (L.) Murr. has demonstrated an array of biological activities that are generally attributed to the presence of spilanthol and other alkylamides. Recently this plant has been of interest due to its potential for the treatment and prevention of malaria. OBJECTIVE The aim of this study was to develop a liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-esiMS) method for rapid identification and quantification of the alkylamide spilanthol from S. acmella. METHODS Hydroethanolic extracts were prepared from fresh S. acmella using different percentages of ethanol and were stored at -80, -20 and 25°C. Spilanthol was isolated and used as a standard for quantitative analysis. RESULTS Validation parameters for the HPLC-esiMS analysis of spilanthol were as follows: repeatability, ≤ 6%; intermediate precision, ≤ 2%; range, 0.45-450 µm; limit of detection, 0.27 µm; and limit of quantification, 0.45 µm. Eight alkylamides in the S. acmella extract were identified based on MS-MS fragmentation patterns, and NMR analysis confirmed the identity of the most abundant of these as spilanthol. Spilanthol was extracted most efficiently in solvents containing >75% ethanol, and was stable in ethanolic extracts stored at all three temperatures. CONCLUSIONS These results demonstrate the effectiveness of HPLC-esiMS for quantitative and qualitative analysis of spilanthol. We show that spilanthol is effectively extracted in ethanol, and is stable in ethanol extracts for over 6 months, even at room temperature.