Sphingolipids generate signals in plants in response to a variety of biotic and abiotic stresses. Measuring these signaling compounds is complicated by the heterogeneity of structures within the sphingolipid family and the comparatively low concentration of their metabolites in plant tissues. To date, the only method with the sensitivity, dynamic range, and specificity to measure all sphingolipids in a plant extract is liquid chromatography coupled to mass spectrometry. The drawback of this method is the cost of the hardware, the expertise in mass spectrometry required to critically assess the outcome and the lack of suitable standards for accurate quantitative analysis. The goal of this chapter is to assist researchers in setting up experiments to measure sphingolipids and explain some of the pitfalls and solutions along the way.