Bioavailability is a quantitative measure of the capacity of a drug to reach systemic circulation. However, if a drug target is localized in a subcellular organelle, then the drug may not be able to reach it and the effect of the drug will not be attained. Although most drug targets are localized within intracellular compartments, specific targeting of drugs at the subcellular level is not well established. Membrane proteins, lipids, nutrients and some pathogens are internalized into the cell to be targeted to distinct subcellular compartments via membrane trafficking. Recent advances have identified novel methods of subcellular drug targeting, involving the use of conjugation to ligands of cell surface receptors or to lipid anchors. In this review, we focus on the importance of subcellular targeting of drugs, in particular, the mechanism of lipid-anchoring as a novel strategy and its potential application for the treatment of several diseases.