Mycobacteria are known for their unique and complex cell wall architecture, characterized by distinct lipid-rich outer layers, making lipid metabolism a central point of their physiological processes. One crucial aspect of all mycobacteria is their ability to metabolize host lipids and to accumulate the resulting neutral lipids in their own cytoplasm in the form of intrabacterial lipid inclusions (ILI). These lipid-rich organelles consist of a hydrophobic core containing neutral lipids, essentially triacylglycerol (TAG), surrounded by a phospholipid monolayer, associated with numerous proteins. These ILI not only serve as reservoirs for energy storage and provide a platform for the synthesis and maintenance of lipids, which are essential for the adaptability and survival of mycobacteria under various environmental stresses. How these mycobacteria acquire host lipids and how ILI are formed remain important and largely unanswered questions. More generally, the anabolism and catabolism of ILI enhance the survival and long-term persistence of mycobacteria and thus represent two major processes controlled by a number of enzymes and regulatory proteins that play an essential role in the biosynthesis, modification, and utilization of these lipid inclusions. In this context, we report here the synthesis of Oxadiazolone (OX) and the Cyclophostin & Cyclipostins (CyC) activity-based probes, and their use for the direct capture of target proteins in M. abscessus growing under carbon excess and nitrogen-deprived in vitro conditions that promote TAG production and ILI-positive phenotype, via bio-orthogonal click-chemistry activity-based protein profiling (CC-ABPP). This approach led to the identification of a set of 65 enzymes potentially involved in the global processes related to ILI anabolism. Among these enzymes, the long-chain-fatty-acid--CoA ligase MAB_1978c/FadD15 has been validated not only as a pivotal enzyme colocalized on ILI, but above all as a major contributor in ILI formation in M. abscessus.