Triacylglycerols (TGs) from plant oils and animal fats are an important part of human diet due to their high nutritional value.TGs are characterized by the total carbon number (CN) and the number, position and configuration (cis-/trans-) of double bonds (DBs) in FA acyl chains.TGs can also differ in the stereospecific positions of FAs on the glycerol skeleton (sn-1, 2 or 3) yielding TG regioisomers.The combination of different FAs and above mentioned types of isomerism in TGs lead to an enormous number of TG species in natural samples.The determination of stereospecific positions of FAs in TGs is important due to the stereospecific environment in the human organism.The characterization of TGs in complex natural mixtures including the determination of different types of isomerism is a challenging task requiring the combination of various separation modes, careful optimization of separation conditions and the use of appropriate detection technique.