A method was developed to separate, identify, and quantify 28 fatty acids of potential health relevance using liquid chromatography/electrospray ionizationmass spectrometry (LC/ESI-MS). Optimization of the experimental factors enabled baseline separation of the fatty acids including three pairs of closely related fatty acid isomers (C18:3n-3 and C18:3n-6; C18:1 and C18:1 t; and C20:3n-3 and C20:3n-6) that are challenging to separate. The limits of detection ranged from 0.01 to 0.26 mg L −1 for the 28 fatty acids, and average recovery (mean, n =4) was found to be 102 ±12 %. In addition, the proposed method was validated using a quality control standard mix of fatty acids which yielded acceptable precision and accuracy. Fatty acid concentrations in conventional grain-fed and organic grass-fed beef were determined, and the results show that grass-fed beef have a lower omega-6 to omega-3 ratio (1.6-2.8) compared to grain-fed beef (9.3-13.5). Principal component analysis (PCA) was applied on the resulting data to find correlations between significant fatty acid composition and the diet of beef samples.