The utilization of 5-aminopropargyl nucleotide serves as a versatile molecular biology tool for the introduction of functional groups into a nucleic acid target of interest by using in-vitro enzymatic incorporation method. This article describes a simple, reliable, general, and efficient two-step chemical method for the synthesis of 5-(3-aminopropargyl)-2'-deoxycytidine-5'-O-triphosphate, 5-(3-aminopropargyl)-cytidine-5'-O-triphosphate, 5-(3-aminopropargyl)-2'-deoxyuridine-5'-O-triphosphate, and 5-(3-aminopropargyl)-uridine-5'-O-triphosphate, starting from the corresponding pyrimidine triphosphate. The first step involves regioselective C-5 iodination of pyrimidine triphosphate using N-iodosuccinimide and sodium azide. In the second step, propargylamine is coupled to the iodo-pyrimidine using the palladium-catalyzed Sonogashira reaction, producing good yields of highly pure (>99.5% HPLC) 5-aminopropargyl-pyrimidine-5'-O-triphosphate. In this approach, the palladium-catalyzed Sonogashira coupling reaction is highly chemoselective and does not involve protection and deprotection. © 2019 by John Wiley & Sons, Inc.