Squalene is a natural biologically active compound. It has a unique pharmacological activity. This isoprenoid is actively involved in metabolism, primarily as a predecessor of biosynthesis of steroids, as a basic structural fragment of the epidermis. This compound is characterized by proliferative, immune-supportive and antioxidant activity. Squalene is also currently being considered as a promising molecule in oncology. Until recently, squalene was extracted from the liver of deep-sea fish, so it was a very expensive product. However, due to the introduction of deep-sea animals catch limits in the world, now this method has lost its relevance. Therefore, it becomes necessary to use other products containing this molecule. Fatty oils of vegetable origin can be possible sources of squalene. The search for unique and most useful fatty oils, including potential sources of squalene, still remains a challenge. As a result of the study, the authors have developed a technique of detection of squalene in vegetable fatty oils by the method of gas chromatography-mass spectrometry. The determination was carried out in the isothermal mode elution evaporator at 270°C, that ensured the desorption of the sample of vegetable fatty oil from the evaporator. The apparent advantage of the proposed method is that the determination of squalene does not require its preliminary extraction and purification of fatty oils. The use of the absolute calibration method enabled to quantify the content of squalene in the studied fatty oils. It was found that the greatest amount of squalene was found in amaranth and pumpkin seed oil.