This study presents a comprehensive global analysis of the yeast lipidome using quantitative shotgun mass spectrometry. The researchers used Saccharomyces cerevisiae as a model organism to demonstrate that automated lipidomics analysis enables the absolute quantification of individual molecular lipid species by processing a single sample of only 2 million yeast cells. They achieved absolute quantification of 250 molecular lipid species covering 21 major lipid classes, achieving approximately 95% coverage of the yeast lipidome with 125-fold improvement in sensitivity compared to previous methods. Comparative lipidomics showed that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches. The lipidome of eukaryotic cells consists of hundreds to thousands of individual lipid species that constitute membranes, store metabolic energy and function as bioactive molecules. Despite the extensive characterization of proteins, their association into complexes and activities, it is still difficult to assess how perturbations within the lipid metabolic network affect the full lipidome of cells. This work shows that lipidome-wide quantification of individual molecular lipid species by absolute quantification provides a new approach to relate lipidomics and functional genomics studies. The yeast Saccharomyces cerevisiae serves as a prime model organism for studying the molecular organization and regulatory circuitry of eukaryotic lipidomes. It uses a relatively simple and conserved network of lipid metabolic pathways that synthesize a few hundred molecular lipid species constituting its full lipidome. The lipidome diversity is primarily determined by the fatty acid synthase, the Δ-9 desaturase, and the fatty acid elongation complex that produce only saturated or mono-unsaturated fatty acids having 10 to 26 carbon atoms for the biosynthesis of glycerolipids, glycerophospholipids, and sphingolipids. Importantly, several metabolic conversions interlink sphingolipid, glycerophospholipid, and glycerolipid metabolism such that any perturbation within the metabolic network is prone to induce lipidome-wide ripple effects. Despite remarkable methodological advances, lipidomics seldom complements functional genomics efforts owing to three major factors. First, analysis of glycerophospholipids and sphingolipids requires two separate lipid extractions, which use vast amounts of sample. Second, the lack of appropriate internal lipid standards limits the quantification scope to profiling analysis or relative quantification. Third, the present methods are not designed for full characterization of molecular lipid species but instead identify 35 to 70 lipids with different sum compositions. Here, the researchers present a mass spectrometric approach that enabled absolute quantification of 250 molecular lipid species constituting 21 lipid classesThis study presents a comprehensive global analysis of the yeast lipidome using quantitative shotgun mass spectrometry. The researchers used Saccharomyces cerevisiae as a model organism to demonstrate that automated lipidomics analysis enables the absolute quantification of individual molecular lipid species by processing a single sample of only 2 million yeast cells. They achieved absolute quantification of 250 molecular lipid species covering 21 major lipid classes, achieving approximately 95% coverage of the yeast lipidome with 125-fold improvement in sensitivity compared to previous methods. Comparative lipidomics showed that growth temperature and defects in lipid biosynthesis induce ripple effects throughout the molecular composition of the yeast lipidome. This work serves as a resource for molecular characterization of eukaryotic lipidomes and establishes shotgun lipidomics as a powerful platform for complementing biochemical studies and other systems-level approaches. The lipidome of eukaryotic cells consists of hundreds to thousands of individual lipid species that constitute membranes, store metabolic energy and function as bioactive molecules. Despite the extensive characterization of proteins, their association into complexes and activities, it is still difficult to assess how perturbations within the lipid metabolic network affect the full lipidome of cells. This work shows that lipidome-wide quantification of individual molecular lipid species by absolute quantification provides a new approach to relate lipidomics and functional genomics studies. The yeast Saccharomyces cerevisiae serves as a prime model organism for studying the molecular organization and regulatory circuitry of eukaryotic lipidomes. It uses a relatively simple and conserved network of lipid metabolic pathways that synthesize a few hundred molecular lipid species constituting its full lipidome. The lipidome diversity is primarily determined by the fatty acid synthase, the Δ-9 desaturase, and the fatty acid elongation complex that produce only saturated or mono-unsaturated fatty acids having 10 to 26 carbon atoms for the biosynthesis of glycerolipids, glycerophospholipids, and sphingolipids. Importantly, several metabolic conversions interlink sphingolipid, glycerophospholipid, and glycerolipid metabolism such that any perturbation within the metabolic network is prone to induce lipidome-wide ripple effects. Despite remarkable methodological advances, lipidomics seldom complements functional genomics efforts owing to three major factors. First, analysis of glycerophospholipids and sphingolipids requires two separate lipid extractions, which use vast amounts of sample. Second, the lack of appropriate internal lipid standards limits the quantification scope to profiling analysis or relative quantification. Third, the present methods are not designed for full characterization of molecular lipid species but instead identify 35 to 70 lipids with different sum compositions. Here, the researchers present a mass spectrometric approach that enabled absolute quantification of 250 molecular lipid species constituting 21 lipid classes