Although the outer membrane of the nuclear envelope is continuous with the endoplasmic reticulum, temporally regulated and functionally significant differences in membrane lipid composition may exist between the two nuclear membranes and between the outer nuclear membrane and the endoplasmic reticulum. Biochemical approaches to probing lipid composition are challenged when lipid dynamics must be analyzed with fine spatiotemporal resolution, for instance, within the cell cycle of a single cell. Here we describe a method to probe the distribution of phosphatidic acid and diacylglycerol, two interconvertible biosynthetic precursors for other membrane glycerophospholipids, in living cells of the model fission yeast Schizosaccharomyces pombe. We show how genetically encoded fluorescent biosensors can be constructed and optimized and present a protocol to probe and quantify phosphatidic acid and diacylglycerol levels specifically at the inner nuclear membrane.