In Mycobacterium tuberculosis a rhamnosyltransferase (WbbL) catalyses the transfer of an alpha-L-Rhap residue from dTDP-L-rhamnose (dTDP-Rha) to decaprenyldiphosphoryl-alpha-D-N-acetylglucosamine (GlcNAc-P-P-DP) to form alpha-L-Rhap-(1-->3)-alpha-D-GlcNAc-P-P-DP, which is then further elongated with Galf and Araf units, and finally mycolylated and attached to the peptidoglycan. This enzyme is essential for M. tuberculosis viability and at the same time absent in eukaryotic cells, and is therefore a good target for the development of new antituberculosis therapeutics. Here, we report a microtitre plate-based method for the assay of this enzyme using a crude membrane preparation from an Escherichia coli strain overexpressing wbbL as an enzyme source and the natural acceptor substrate GlcNAc-P-P-DP. Initial characterization of the enzyme included unequivocal identification of the product Rha-GlcNAc-P-P-DP by liquid chromatography (LC)-MS, and the facts that WbbL shows an absolute requirement for divalent cations and that its activity is stimulated by beta-mercaptoethanol. Its pH optimum and basic kinetic parameters were also determined, and the kinetic analysis showed that WbbL uses a ternary complex mechanism. The microtitre plate-based assay for this enzyme was developed by taking advantage of the lipophilic nature of the product. This assay should be readily transferable to other glycosyltransferases which use lipid-based acceptors and aid greatly in obtaining inhibitors of such glycosyltransferases for new drug development.