A simple and sensitive method for the determination of free fatty acids (FFAs) using acridone€́9€́ethyl€́p€́toluenesulfonate (AETS) as a fluorescence derivatization reagent by high performance liquid chromatography (HPLC) has been developed. Free fatty acid derivatives were separated on an Eclipse XDB€́C₈ column with a good baseline resolution and detected with the fluorescence of which excitation and emission wavelengths of derivatives were set at _ex=404 and _em=440 nm, respectively. Identification of 19 fatty acid derivatives was carried out by online post€́column mass spectrometry with an atmospheric pressure chemical ionization (APCI) source under positive€́ion detection mode. Nineteen FFAs from the extract of <i>Lomatogonium rotatum</i> are sensitively determined. The results indicate that the plant <i>Lomatogonium rotatum</i> is enriched with an abundance of FFAs and FFAs of higher contents, which mainly focus on even carbon atoms, C₁₄, C₁₆, and C₁₈. The validation of the method including linearity, repeatability, and detection limits was examined. Most linear correlation coefficients for fatty acid derivatives are >0.9989, and detection limits (at signal€́to€́noise of 3:1) are 12.3-43.7 fmol. The relative standard deviations (RSDs) of the peak areas and retention times for 19 FFAs standards are <2.24% and 0.45%, respectively. The established method is rapid and reproducible for the separation determination of FFAs from the extract of <i>Lomatogonium rotatum</i> with satisfactory results.