Inhibition of the MDM2-p53 interaction can stabilize the p53 protein and offer a novel strategy for cancer therapy. The imidazoline compound (Nutlin-3) is a promising small molecule antagonist of the MDM2-p53 interaction. This compound was synthesized as a racemic mixture, and one enantiomer is 100-200-fold more active than the other enantiomer. In this study, various enantiomeric separation approaches were explored to resolve the Nutlin-3 enantiomers using chiral supercritical fluid chromatography (SFC) as well as chiral liquid chromatography (LC) under normal phase mode, reversed phase mode and polar organic phase mode. The chiral SFC method based on Chiralcel OD column showed superior separation in terms of selectivity and efficiency. Optimization of the chiral separation method enabled high throughput preparative scale purification. Ultimately, 5 g of racemic mixture were purified on Prep-SFC in 75 min with the recovery rate above 92%.