Mammalian fatty acid synthase (FASN) is a lipogenic enzyme that catalyzes the formation of the long chain saturated fatty acid palmitate from acetyl and malonyl CoA in the presence of NADPH. Mammalian cells acquire fatty acids through dietary sources or through FASN. Although most mammalian cells express FASN at low levels, it is upregulated in cancers and during replication of many viruses. The precise role of FASN in disease pathogenesis is poorly understood, and whether de novo fatty acid synthesis contributes to host or viral protein acylation has been traditionally difficult to study. We describe a cell permeable, click-chemistry compatible alkynyl-acetate analog (5-Hexynoic acid, or "Alk-4") that functions as a reporter of FASN-dependent protein acylation. Alk-4 metabolic labeling enabled biotin-based purification and identification of more than 200 FASN-dependent acylated cellular proteins. Alk-4 also labeled the palmitoylated host protein IFITM3 (Interferon inducible transmembrane protein-3), a restriction factor for Influenza, and the myristoylated HIV-1 MA (Matrix) protein. Thus, Alk-4 is a useful bioorthogonal tool to selectively probe FASN-mediated protein acylation in normal and diseased states.