In the current study, the effect of ghee extraction methods (direct cream DC, milk butter MB and milk skin MS) on its molecular composition has been investigated using Fluorescence spectroscopy. The excitation wavelength of 300 nm was found the best to produce pronounced spectral signatures of beta-carotene, vitamins and conjugated linoleic acid (CLA) in both cow and buffalo ghee types. Principal component analysis (PCA) has been applied on the spectral data to visualize the classification among ghee samples extracted by three methods. Both cow and buffalo ghee contain spectral signatures of vitamin A, E, K, D and CLA which has been verified through plotting loading vectors. The analysis of loading plots has been suggested that for cow ghee, MS extraction method conserve relatively higher concentration of beta carotene while DC and MB methods are a good choice for preserving relatively more concentrations of vitamins D, E and K. Similarly, for buffalo ghee, MS extraction method appear with higher concentration of CLA, whereas DC extraction method looks to preserve relatively higher concentration of vitamin A while MB method retains relatively low concentration of CLA and vitamins as compared to other two methods.