In this study, the potential of Raman spectroscopy has been utilized to characterize the methods direct cream (DC), milk butter (MB) and milk skin (MS) used for the extraction of desi ghee from buffalo and cow milk. Raman spectra from six types of ghee samples extracted by above methods were acquired using two laser wavelengths of 532 and 785 nm. The Raman spectra of cow ghee revealed that it contains three bands of beta-carotene at 1005, 1156 and 1520 cm−1 which differentiated it from buffalo ghee. To highlight small spectral differences, statistical analysis through principal component analysis (PCA) has been performed on the Raman spectra of ghee samples to reach subsequent conclusion. Based on the variations in molecular composition of cow ghee samples, it has been found that DC method retain relatively higher concentration of beta-carotene and MB method contain higher concentration of conjugated linoleic acid (CLA) and fatty acids than MS method. Similarly, DC & MS methods were found best for retaining relatively higher concentration of CLA and fatty acids in buffalo ghee as compared to MB method which retains relatively higher concentration of fatty acids. Display omitted •Desi ghee is a type of clarified butter obtained from buffalo and cow milk.•Desi ghee is commonly extracted by Direct cream (DC), Milk butter (MB) and milk skin (MS) methods.•Both ghee types contained valuable ingredients which slightly change their concentration during extraction methods.•DC method retains higher concentration of beta-carotene and MB was found to retain higher concentration of CLA, lipids and fatty acids in cow ghee.•Buffalo ghee extracted from DC & MS method retain higher concentration of CLA and MB method retain slightly higher concentration of fatty acids and lipids.