Lipid second messengers play important roles in many cell signaling cascades. Lipid signaling molecules allow for high specificity, rapid transduction, and rapid reversibility of localized stimulation events. Fluorescent sensors capable of detecting individual signaling lipids enable their production and degradation to be followed, revealing the nature and dynamics of signaling pathways. The following sections outline a method for using lipid sensors to monitor the production of signaling lipids on the plasma membrane of C2C12 myotubes in response to insulin signaling.