In this study, an optimized vortex-assisted method for rapid screening of oil content in Jatropha curcas L. (Jatropha) seeds is described. A 24-1IV fractional factorial design was employed to study the influence of vortex stirring rate, sample:solvent ratio, extraction time, and the number of extraction cycles in the Jatropha oil extraction efficiency. Gas chromatography-mass spectrometry (GC-MS) was used for the identification of the fatty acids. The number of extraction cycles was the most important factor, and the optimum conditions were two extractions with hexane at a sample:solvent ratio of 1:5 mg:µL, under vortex-stirring at 2500 rpm for 1 min. Linoleic acid, oleic acid, and palmitic acid were the major fatty acids of Jatropha oil. The method showed inherent advantages of simplicity, accuracy, short time of analysis, and low consumption of both organic solvent and sample, and may serve as a cost-effective alternative to the modified Soxhlet method in estimation of oil content and composition in Jatropha breeding programs.