The fermentation of corn into ethanol is a major usage of the American-grown corn crop. During the process of corn to ethanol, corn lipid class composition changes. The formation of free fatty acid (FFA) from triacylglycerol is commonly observed. Increased free fatty acid fermentation byproducts result in troublesome processing to remove excess FFA. The objective of this work was to determine where in the process of corn to ethanol FFA is generated using both industrial and laboratory-scale approaches. Specific commercial steps and conditions in the process of corn to ethanol were tested to determine their role in the generation of FFA. Thin-layer chromatography (TLC) and gas chromatography (GC) were used to quantify FFA in extracted total lipids. FFA assay by titration was also used for quantification to compare to TLC-based methods. Results indicate generation may be due to incremental increases at each step in the process, not due to one specific processing step. Furthermore, laboratory-based fermentations indicate the possible oxidation of linoleic acid, particularly in the FFA fraction, in corn may increase % FFA determined using titration-based methods. Industrial corn oil samples were tested and shown to be oxidized leading to an overestimate of FFA by 15-25% using FFA assay via titration compared to TLC-GC methods due to acid formation. Overall FFA generation is seen throughout the entire process of corn to ethanol, and this trend also agrees with the increased FFA levels in industrial samples observed in multiple ethanol plants and studies.