The regulatory pathways required to maintain eukaryotic lipid homeostasis are largely unknown. We developed a systematic approach to uncover new players in the regulation of lipid homeostasis. Through an unbiased mass spectrometry-based lipidomic screening, we quantified hundreds of lipid species, including glycerophospholipids, sphingolipids, and sterols, from a collection of 129 mutants in protein kinase and phosphatase genes of Saccharomyces cerevisiae. Our approach successfully identified known kinases involved in lipid homeostasis and uncovered new ones. By clustering analysis, we found connections between nutrient-sensing pathways and regulation of glycerophospholipids. Deletion of members of glucose- and nitrogen-sensing pathways showed reciprocal changes in glycerophospholipid acyl chain lengths. We also found several new candidates for the regulation of sphingolipid homeostasis, including a connection between inositol pyrophosphate metabolism and complex sphingolipid homeostasis through transcriptional regulation of AUR1 and SUR1. This robust, systematic lipidomic approach constitutes a rich, new source of biological information and can be used to identify novel gene associations and function.