This article discusses the methods most commonly employed in the analysis of the triacylglycerols (TAGs) in natural fats and considers the main advantages and disadvantages of each and the techniques for optimising analytical conditions. Complete analysis of the composition of a natural fat calls for a method of extracting and purifying the triglyceride fraction, normally by preparatory thin-layer and column chromatography. Determination of the individual components of triglyceride mixtures still entails certain difficulties, namely, the separation and identification of the TAGs in natural fats. High-performance liquid chromatography (HPLC) offers significant advantages over gas and thin-layer chromatography. Many workers have developed non-aqueous, reversed-phase HPLC systems capable of successfully resolving complex mixtures of TAGs, and combining reversed-phase (RP) HPLC and argentation chromatography may improve the results. Identification of the TAGs separated by HPLC becomes an extremely complex task if many different fatty acids are involved and if the sn-stereoscopic positions on the glycerol are to be determined. Enzymatic analysis and chiral-phase chromatography are capable of localising fatty acids on the TAG molecule. In closing, some of the most interesting biomedical applications of TAG analysis are summarised.