Three isomers of aminopyridine separate well by reversed-phase cation-exchange mixed-mode chromatography on an Obelisc R HPLC column. The presence of additional mechanism of interaction provides great selectivity for separation of closely related compounds. Elution of compounds can be monitored by UV, Evaporative Light-Scattering Detector (ELSD), Corona (CAD) or LC/MS. This method was validated at a pharmaceutical company. This HPLC method can be adopted as general approach for analysis of aminopyridine and other isomeric compounds.