This study presents a comprehensive and quantitative analysis of the yeast lipidome using shotgun lipidomics. The authors developed a method that enabled the absolute quantification of 250 molecular lipid species across 21 major lipid classes, achieving a 125-fold improvement in sensitivity compared to previous methods. This approach required only 2 million yeast cells, significantly reducing the sample size compared to traditional methods. The study found that changes in growth temperature and defects in lipid biosynthesis machinery can lead to widespread alterations in the yeast lipidome, demonstrating the importance of lipidomics in understanding the functional genomics of eukaryotic organisms. The results provide a detailed resource for further studies on lipid metabolism and membrane trafficking in yeast.This study presents a comprehensive and quantitative analysis of the yeast lipidome using shotgun lipidomics. The authors developed a method that enabled the absolute quantification of 250 molecular lipid species across 21 major lipid classes, achieving a 125-fold improvement in sensitivity compared to previous methods. This approach required only 2 million yeast cells, significantly reducing the sample size compared to traditional methods. The study found that changes in growth temperature and defects in lipid biosynthesis machinery can lead to widespread alterations in the yeast lipidome, demonstrating the importance of lipidomics in understanding the functional genomics of eukaryotic organisms. The results provide a detailed resource for further studies on lipid metabolism and membrane trafficking in yeast.