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Major constituents of all living beings are nano-sized molecular species like lipids, proteins, fatty acids, and nucleic acids which are constantly in communication with each other through a complex interwoven molecular network. Understanding a cellular physiological function at the molecular level requires differentiation of the self-assembled biomolecules. Photo physical studies of 8-anilino-1-napthalenesulphonate (ANS) and Nile red (NR) are used to explore differentiation of these biomolecules. An efficient Förster resonance energy transfer (FRET) from ANS to NR causes significant fluorescence quenching of ANS in lipid, whereas it becomes practically insignificant for protein. From the present study, FRET efficiency appears as a marker for differentiating these self-assembled macromolecules. To this end, a novel physical method based on FRET efficiency has been developed to measure the total lipid content of milk.