The separation of pramipexole enantiomers using polysaccharide-based chiral stationary phase (CSP) columns with organic-aqueous mobile phases was reported for the first time. The (R)-enantiomer was separated from pramipexole using a Lux®Cellulose-1 column with a mobile phase consisting of solvent A (acetonitrile):solvent B (0.1% diethylamine with 20 mM ammonium bicarbonate) (30:70, v/v). The elution time was about 7.0 min and the resolution between the enantiomers was greater than 8.0. The linear range of the method was 0.3–3.6 μg mL−1with the limit of detection and limit of quantification for the (R)-enantiomer were 0.1 µg mL−1and 0.3 µg mL−1, respectively. The percentage recovery of (R)-enantiomer ranged from 97.1 to 100.1 for tablet dosage forms of pramipexole dihydrochloride monohydrate. In this study, the enantiomeric separation of pramipexole was achieved. The results demonstrated good linearity, precision, and accuracy for quantifying (R)-enantiomer in tablet dosage forms, suggesting the applicability of a quantitative method that uses polysaccharide-based CSPs in combination with organic-aqueous mobile phases.