Depending on their chemical nature, lipids can be classified in two main categories: hydrophilic, greatly contributing to membrane composition and subcellular organelle compartmentalization, and hydrophobic, mostly triglycerides, greatly enrolled in the storage and production of energy. In both cases, some lipid molecules can be involved as signaling agents in the regulation of metabolism and protective or damaging pathways in responses to harmful stimuli. These events could affect in particular the liver, because of its central role in the maintenance of lipid homeostasis. Lipids have been demonstrated to fluoresce, contributing to the overall emission signal of the liver tissue along with other endogenous fluorophores, relatable to energy metabolism and oxidative events. The mere estimation of the fluorescing lipid fraction in parallel with the other endogenous fluorophores, and with the common biochemical and histochemical biomarkers of tissue injury has been exploited to investigate the liver morpho-functional conditions in experimental hepatology. More interestingly, the fluorescing lipid fraction is greatly relatable to free fatty acids such as arachidonic, linoleic and linolenic acid, which are deserving increasing attention as precursors of products involved in several and complex signaling pathways. On these bases, the ability of autofluorescence to detect directly arachidonic acid and its balance with other unsaturated fatty acids may be exploited in the diagnosis and follow-up of fatty livers, helping to improve the personalization of the metabolic/lipidomic profiling. This could also contribute to elucidate the role of the injuring factors in the choice of suitable donors, and in the set-up of preservation procedures in liver transplantation.