cis-Vaccenate was determined in milkweed (Asclepias) seeds employing supercritical fluid extraction (SFE) with in-situ enzymatic methanolysis. Candida antarctica lipase type B immobilized onto two different types of carrier materials-a hydrophobic polymer (Novozyme 435) and a silica-based material (NovoSample 40013)-was investigated. Several SFE parameters were investigated to find the optimal conditions, including extraction time, temperature, methanol concentration, and water level. It was shown that 90 min of dynamic extraction/reaction time was required, and that the optimal methanol and water concentrations differed for the two enzyme preparations studied. Overall, Novozyme 435 demonstrated the fastest reaction kinetics and consistently gave the highest vaccenate yields. The optimized methodology was applied on 15 species of milkweed seeds, giving an average recovery of 105 ± 7% when compared to results obtained using a conventional methodology. The average relative standard deviations were 8% for the enzyme-based method and 4% for the reference method. The proposed methodology was faster, was less laborious, and consumed less organic solvent than the reference method. Hence, SFE with in-situ enzymatic methanolysis is a promising methodology for the analysis of FA in oilseeds.