Ion-pairing reversed-phase liquid chromatography (RPLC) was used to separate two polysulfonates, rutin nona(H-) sulfonate sodium and rutin deca(H-) sulfonate sodium, which have very similar chemical structures. The final product always contained both of them when one of the compounds was synthesized. Baseline separation was achieved on a C8-bonded silica column at ambient temperature. The eluent was acetonitrile–15mMphosphate buffer solution containing 20mMTBA(pH 6.0) (46:54, v/v). The calibration plotwas linear in the concentration range 0.5–200μgml−1 for both analytes. The limits of detection (LODs; 254nm) were 0.03μgml−1 for rutin nona(H-) sulfonate sodium and 0.04μgml−1 for rutin deca(H-) sulfonate sodium. Three batches of rutin deca(H-) sulfonate sodium were analyzed using the assay; the results showed that the analytical performance is really satisfactory.