Rab GTPases represent a family of key membrane traffic regulators in eukaryotic cells. To exert their function, Rab proteins must be modified with one or two geranylgeranyl moieties. This modification enables them to reversibly associate with intracellular membranes. In vivo the newly synthesized Rab proteins are recruited by Rab escort protein (REP) that presents them to the Rab geranylgeranyl transferase (Rab GGTase), which transfers one or two geranylgeranyl moieties to the C-terminal cysteines. Detailed understanding of the mechanism of prenylation reaction and subsequent membrane delivery of Rab proteins to the target membranes were hampered by lack of efficient technologies for the generation of preparative amounts of prenylated Rab GTPases. To circumvent this problem, we developed an approach that combines recombinant protein production, chemical synthesis of lipidated peptides with precisely designed and readily alterable structures, and a technique for peptide-to-protein ligation. Using this approach, we generated a number of semisynthetic prenylated Rab GTPases. Some of the proteins were also supplemented with fluorophores, which enabled us to develop a fluorescence-based in vitro prenylation assay. The approach described allows production of preparative amounts of prenylated GTPases, which was demonstrated by generation and crystallization of a monoprenylated YPT1:Rab GDI complex.