Low-density dispersive liquid-liquid microextraction with a subsequent esterification step was proposed in this study for the determination of linoleic acid and stearic acid in sheep blood serum samples. The method developed aimed to quickly and efficiently extract and esterify both fatty acids with subsequent analysis by gas chromatography/flame ionization detection. The extraction method was optimized with Scheffé’s polynomial model for three-component mixtures. The method was validated according to the parameters established by Eurachem Guide (2014). The optimal extraction conditions for LD-DLLME were 1400 µL of dispersion medium (MgCl2 0.017%), 400 µL of extractor solvent (toluene) and 1200 µL of dispersion solvent (methanol). The method performance showed adequate selectivity, sensitivity and precision to be applied to real samples, with an average recovery of 98.54% for linoleic acid and 103.83% for stearic acid. LD-DLLME was superior to the traditional method of analysis, which has been used until now for the determination of fatty acids in blood serum samples from ruminants. The analysis of real samples showed that the developed method is efficient for monitoring these substances in ruminants. Lowdensity Low density liquidliquid liquid chromatographyflame chromatography flame detection Scheffés Scheffé s threecomponent three component mixtures 2014. 2014 . (2014) LDDLLME LD DLLME 140 MgCl2 MgCl (MgCl 0.017%, 0017 0.017% , 0 017 0.017%) 40 toluene (toluene 120 methanol. methanol (methanol) selectivity 9854 98 54 98.54 10383 103 83 103.83 ruminants 201 (2014 14 001 0.017 01 4 12 (methanol 985 9 5 98.5 1038 10 8 103.8 20 (201 1 00 0.01 98. 103. 2 (20 0.0 (2 0. (