Direct transesterification (DT) and solvent extraction with acid or basic derivatization procedure for fatty acid (FA) analysis in herbage were compared. The highest total FA, alpha-linolenic and linoleic acid contents were obtained with DT. However, DT also produced the highest amount of interfering compounds, identified as phytadienes and sugar derivative products, which may co-elute with FA. An additional step based on solid-phase extraction to produce clean samples was proposed. This procedure is simple and gives good recoveries for the FA fortified samples. Additionally, structural characterization of 16:1 trans-3 was conducted by covalent adduct chemical ionization tandem mass spectrometry.