Trans-fatty acids enter the human body with hydrogenated fats. The oil and fat industry must inform consumers about the fatty acid composition of food products, including the content of saturated fatty acids and trans-isomers. This study used the method of instrumental analysis to determine the fatty-acid profile and the content of trans-fatty acids in margarine. The research involved ten commercial samples of margarine. The fatty acid composition was studied by gas chromatography and mass spectrometry. The calibration graphs were based on standard mixes of methyl esters of individual fatty acids. Some oils appeared to contain ≥ 17% of palmitic acid. The total content of saturated fatty acids was 20.04–38.84%; the content of monounsaturated acids ranged from 27.92 to 36.81%, while that of polyunsaturated acids was between 14.51 and 28.99%. The margarine samples contained no butter (dairy) fat. All the samples contained 0.01–23.06% of trans-fatty acids. If the share of trans-fats exceeded 2%, it meant that the technical regulations had been violated, and hydrogenated oils had been introduced into the formulations. The highly specific mass spectrometric approach made it possible to detect trace trans-fatty acids, thus eliminating the chance of unreliable or false positive results. These methods proved to be an effective means of regulatory compliance and counterfeit prevention.