Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner. The roles of lipids in cells go far beyond providing the structural backbone of cellular membranes. Certain lipid species are powerful signaling molecules. Examples include the roles of sphingosine (Sph) and the diacylglycerol (DAG) variant, stearoyl-arachidonylglycerol (SAG) in intracellular calcium signaling (1, 2). The study of such signaling lipids is often complicated by the fact that they are under tight metabolic control and that they occur only in very low concentrations. Overexpression of metabolic enzymes for manipulation of signaling lipid levels is a slow process compared with the rapid turnover of those lipids and may therefore produce not only the target lipid but also multiple downstream metabolites. Chemical dimerizer and optogenetic approaches are options to manipulate lipid contents more rapidly, but they depend on cytosolic lipid-metabolizing enzymes. In the past, many applications therefore focused on phosphoinositides (3, 4). A more general way to rapidly increase lipid concentration is the use of caged lipids. These are equipped with a photocleavable protecting group (caging group), which blocks biological activity and renders them resistant to metabolic turnover before the active lipid is released using a flash of light (2, 5⇓–7). The sudden increase in target lipid concentration facilitates analysis of downstream lipid signaling events as well as lipid metabolism within living cells in pulse−chase experiments. To correctly interpret such signaling events, underlying processes such as lipid−protein interactions, intracellular lipid localization, and kinetics of lipid metabolism need to be considered. To date, lipid metabolism is typically monitored using isotope-labeled or alkyne-modified lipids (8⇓–10). Fluorescent lipids, lipid-binding antibodies, or lipid biosensors are mainly used to study lipid localization (11, 12). Most assays for studying lipid−protein interactions rely on reconstituted membranes/liposomes and are therefore largely restricted to soluble proteins (13⇓⇓–16). The plethora of methods used to investigate these different processes makes it difficult to compare or validate their respective results. A promising approach to integrate the study of lipid metabolism, lipid localization, and lipid−protein interactions has emerged in recent years; bifunctional lipids feature a small diazirine group to allow photo–cross-linking with interacting proteins in the intact cellular environment and a terminal alkyne for subsequent functionalization (17). Biotinylation of cross-linked lipid−protein conjugates enables their enrichment and identification of lipid-interacting proteins. To date, bifunctional lipids are one of the few methods to screen for lipid−protein interactions in living cells (18⇓⇓–21). Alternatively, bifunctional lipids can be used to visualize lipid localization by click reaction with a fluorophore (1, 18, 20). The application of the bifunctional lipid principle to signaling lipids, however, is handicapped by their tight metabolic control. Any precursor is rapidly incorporated into downstream lipids, complicating the interpretation of resulting data. The ability to liberate a single, well-defined signaling lipid species within cells and to immediately capture its interacting partners, investigate downstream signaling, and study its subcellular localization would enable much-needed insight into the regulation of lipid-dependent signaling. Here, we present “trifunctional” lipids as tools, combining the advantages of caged and bifunctional lipids in a single molecule to allow for a wide range of studies in living cells with tight temporal control. Applied to Sph and DAG, we show that trifunctional lipids enable (i) acute alteration of signaling lipid concentration, (ii) measurement of lipid metabolism on a population-wide as well as on a single-cell level, (iii) screening for lipid−protein interactions, and (iv) direct visualization of lipid localization by light and correlative light and electron microscopy (CLEM) in comparable experimental settings.