Abstract Singly- and dually-labeled synthetic oligonucleotides were purified by ion-pair reversed-phase high-performance liquid chromatography using a 50×4.6-mm column packed with porous, 2.5 μm C 18 sorbent. We studied the mechanism of dye-labeled oligonucleotide retention in order to improve the quality of purification. By-products of oligonucleotide synthesis were characterized by liquid chromatography with mass spectrometry detection (LC–MS). We purified oligonucleotides labeled with 6-carboxyfluorescein (6FAM), hexachlorofluorescein (HEX), tetrachlorofluorescein (TET), carboxytetramethylrhodamine (TAMRA) and indodicarboxycyanine (Cy™3) dyes, as well as dually-labeled TaqMan™ probes. Purification of a 0.1-μmole oligonucleotide synthesis in a single injection was demonstrated.