The aim of the study is to compare the transformation cholesterol standard and cholesterol present in a food matrix (butter) during thermal treatment at 150 and 180 °C for 1 h, which results in the formation of cholesterol oxidation products (0.5–189.3 mg g−1 of unheated standard and 0.044–0.063 mg g−1 of unheated butter) and cholesterol dimers (72.3–120.3 mg g−1 of unheated standard and 0.208 mg g−1 of unheated butter). The presence of cholesta‐3,5‐diene is investigated, especially in free cholesterol and cholesteryl palmitate unheated samples (105.3–116.4 mg g−1), as well as in thermally processed butter (0.009 mg g−1). Moreover, the processes of the extensive degradation of cholesterol are observed in standards samples (34.7–98.8% losses) compared to butter samples (25.5–73.5% losses). The qualitative and quantitative analyses of the above mentioned compounds are performed with the use of solid‐phase extraction, gas chromatography coupled with mass spectrometry and gel permeation chromatography. In conclusion, the thermal processing of butter and products containing cholesterol should be performed at mild temperatures, for example, 150 °C, because fewer negative components (e.g., oxysterols, cholesterol dimers) are formed in those conditions compared to heat processing at higher temperatures.Practical Application: The methods described in this study may be useful for the determination of sterols, oxysterols, cholesterol dimers, and cholesta‐3,5‐diene in standard cholesterol samples and cholesterol present in food products. The presented results data are crucial for the assessment of cholesterol changes during processing, especially the observation that some of the components have adverse effects on the human metabolism of oxysterols or cholesterol dimers. The investigations show that the thermal processing of products containing cholesterol should be performed at the lowest possible temperatures, for example, 150 °C, because this protects cholesterol from dimerization, oxidation, and degradation.Thermal processing of cholesterol can lead to the formation of cholesterol dimers, cholesterol oxidation products or cholestadienes, which can have adverse effect of the human health.