A simple and rapid ultra-performance liquid chromatography-electrospray ionization mass spectrometry method for the simultaneous determination of thirteen free fatty acids (FFAs) in Pheretima has been developed and validated. Measurements for each FFA were linear over a wide range (0.05-3.95 mu g mL(-1)) with good correlation coefficients (> 0.99). The limit of detection and limit of quantification for all the fatty acids were below 26 and 78 ng mL(-1), respectively. The intra- and inter-assay precision and accuracy for the thirteen FFAs fell well within the predefined limits of acceptability. Satisfactory recoveries were in the range of 96-103%.