Fatty acid triglycerides are essential components of the human diet, while possessing unique biological activity. Their content in biological media is traditionally determined by gas and liquid chromatography. They are not readily available for mass analysis of food products and blood serum due to the complexity of these methods. The aim of our work is to study the correlation of the absorption spectra of near-infrared radiation with the characteristic oscillations of functional groups of clinically important fatty acids, such as palmitic saturated ones and oleic and linoleic unsaturated ones, as well as their trans -isomers, as well as to develop techniques and test equipment for rapid assessment of their content in fat products and blood serum. As a result, evidence of the applicability of the Fourier spectrometer in the wavelength range of 1.0–2.4 μm for the operational analysis of clinically important fatty acids in butter, spreads and serum, as well as the correlation of the absorption spectra of serum with the content of total triglycerides and cholesterol were revealed. In addition, using a portable spectrometer in the wavelength range of 1.0–1.65 μm suitable for mass analysis of fat-and-oil products quality, the content of these fatty acids was determined, and the selectivity of their determination was studied.