Chloroquine is a chiral antimalarial drug and demonstrates enantioselective pharmacodynamic and pharmacokinetic properties. However, this drug is administered as racemate. The knowledge of stereoselective aspects of these agents may be useful to better understand their mechanisms of action and to optimize their safety and/or clinical efficacy. In this study, an enantioselective analytical method for the quantification of chloroquine enantiomers was developed using HPLC-UV. The chromatographic conditions were: Chirobiotic V column (100 × 2.1 mm, 5 μm) at 25°C, mobile phase containing methanol:acetic acid:triethylamine (100:0.12:0.12), flow rate 1 mL/min, injection volume 10 μL and detection at 258 nm. The validation parameters evaluated were selectivity, linearity, precision, accuracy, and robustness. In addition, a stability study after forced degradation of chloroquine enantiomers was performed. The enantioseparation of chloroquine using a polysaccharide-based chiral stationary phase (Chiralpak ID) at different mobile phase composition was evaluated and the chromatographic performance of both columns was compared. Thus, a stability-indicating chiral analytical method was developed and fully validated, allowing the separation of chloroquine enantiomers and its degradation products in tablets available in Brazil.