Evaluating lumefantrine enantioselectivity and properly quantifying its enantiomers is essential for a deeper understanding of the impact of the enantioselectivity in the pharmacodynamic and pharmacokinetic properties of the drug. For this, a chiral chromatographic method was developed and validated for the separation of lumefantrine enantiomers. The method developed employed a Chiralpack AD-H column (150 × 4.6 mm, 5 μm), with amylose tris-3,5-dimethylphenylcarbamate stationary phase, at 25 °C, mobile phase composed of hexane and isopropanol (97:3), flow rate of 1.0 mL/min and detection at 335 nm. The method showed adequate resolution for lumefantrine enantiomers and was validated for linearity (48–72 µg/ml for each enantiomer), precision, accuracy, selectivity and robustness. The proposed method was successfully applied for determination of lumefantrine enantiomers in tablet formulations and may be useful to future researchers for chiral evaluation of lumefantrine. Graphic Abstract