This protocol describes the synthesis of a lipid-like molecule carrying a head group containing two nitrilotriacetic acid moieties. This multivalent chelator lipid can be incorporated into lipid membranes, to which histidine-tagged protein can then be tethered in an oriented fashion. Possible applications of this lipid are protein tethering to solid-supported membranes, to lipid vesicles or to live cells. As compared to conventional monovalent chelator lipids, this lipid can achieve highly stable tethering of proteins by the multivalent chelator head. The eight-step synthesis described in this protocol can be completed within 4-5 weeks.