The fatty acid profile of cheese influences its sensory parameters, such as color, texture, or flavor. Examining the fatty acid profile also helps to assess the nutritional value of the cheese that is being tested. However, the determination of fatty acids in cheese samples is a multi-stage and time-consuming task. In addition, large amounts of toxic organic solvents are used to prepare samples for analysis purposes. This paper presents the results of a study to determine the fatty acid profile of yellow cheese samples. Six different methods of sample preparation were compared for analysis purposes. The profile of fatty acids was determined using gas chromatography with flame ionization detection (GC-FID). The study showed significant differences (p> 0.05) in the resulting fatty acid profile between the methods used. It was found that the most reliable fatty acid profile results were obtained using methods derived from the Folch method. In addition, tools such as the Analytical Eco-Scale tool and the Analytical Greenness Metric for Sample Preparation (AGREEprep) tool were used to assess the greenness of the methods used. In the case of the Analytical Eco-Scale tool, all six methods scored ‘acceptable green analysis’ with scores ranging from 61 to 73. However, an evaluation of methods using the AGREEprep metric showed that the results of the methods (0.13–0.27) did not show the “greenness” of the analytical methods.