The regulatory pathways required to maintain eukaryotic lipid homeostasis are still largely unknown, which limits the possibilities of hypothesis driven research. Here, we performed a mass spectrometry-based lipidomic screening of yeast strains knocked out for genes of protein kinases and phosphatases, in search for new regulators of lipid homeostasis. We have uncovered novel regulations of lipid homeostasis, revealing a control of acyl chain length by nutrient sensing pathways, a control of complex sphingolipids by a phosphate sensing pathway and new players in the control of sphingolipid metabolism. We showed that systematic lipidomics is a powerful tool to gain novel insights into lipid homeostasis. Due to close interaction between lipid metabolism and other cellular processes it is clear that many of these new lipidomic data from kinases and phosphatase mutants will facilitate the understanding of signaling networks.