Post-translational S-palmitoylation plays a central role in protein localization, trafficking, stability, aggregation, and cell signaling. Dysregulation of palmi-toylation pathways in cells can alter protein function and is the cause of several diseases. Considering the biological and clinical importance of S-palmitoylation, tools for direct, in vivo modulation of this lipid modification would be extremely valuable. Here, we describe a method for the cleavage of native S-palmitoyl groups from proteins in living cells. Using a cell permeable, cysteine-functionalized amphiphile, we demonstrate the direct depalmitoylation of cellular proteins. We show that amphiphile-mediated depalmitoylation (AMD) can effectively cleave S-palmitoyl groups from the native GTPase HRas and successfully depalmitoylate mislocalized proteins in an infantile neuronal ceroid lipofuscinosis (INCL) disease model. AMD enables direct and facile depalmitoylation of proteins in live cells and has potential therapeutic applications for diseases such as INCL, where native protein thioesterase activity is deficient.