To support labeling, claims, and authenticity of food products, industry needs reliable methods for the analysis of fatty acids, including trans fatty acids (TFA). In finished products, precise quantification of TFA can be problematic due to the occurrence of various positional and geometrical isomers originating from different sources, such as animal fats or processed vegetable oils and fats. The risk of underestimating TFA amounts is particularly high when inappropriate GC conditions are used. Complex sample preparation procedures involving purification of TFA isomers by silver ion chromatography have been well-documented and used for research purposes. However, in the food industry, time and cost constraints do not permit multiple analytical steps; therefore, streamlined methods are necessary. Direct methods include preparation of fatty acid methyl esters directly from food samples without prior extraction. The appropriate resolution is obtained using high-resolution GC with a highly polar 100 m capillary column, and quantification is achieved using experimentally determined response. We found that it is possible to quantify TFA in the range of 0.01 to 5.00 g/100 g of lipids in a wide range of food products. In addition, the use of direct transmethylation, response factors, and high-resolution GC allow accurate quantification of other fatty acids, including polyunsaturated and long-chain polyunsaturated fatty acids.