With the developments in high performance chromatography and mass spectrometry, the analysis of complex mixtures of closely related triacylglycerols has come within reach. Currently, however, the availability of chemically pure triacylglycerols is limited, mainly due to the lack of efficient synthetic methods that allow their synthesis. Here, we report an efficient and operationally simple three-step synthesis of enantiopure triacylglycerols with yields ranging from 79 to 92%, that tolerates a broad range of saturated and unsaturated fatty acids. Furthermore, we developed an automated protocol, which might greatly facilitate the synthesis of triacylglycerol libraries. Practical applications: The analysis of the composition of milk fat and as analytical standards in lipidomics.