The effect of ten water-soluble organic solvents on MEKC separation of palonosetron hydrochloride (PALO) stereoisomers using sodium cholate (SC) as chiral selector has been studied. The first chiral CE method fit for the analysis of unwanted PALO distomers (enantiomeric impurities) of low concentrations in the presence of high concentration of the main eutomer has been developed, based on solvent-modified MEKC mode. It was found that methanol provides the best separation among the solvents tested. And an SC concentration of 30mMis proper to provide good resolutions in shorter time and adequate sample capacity, instead of 70 mM as previously reported. The developed method can be used to analyze unwanted PALO distomers of a few micrograms per milliliter in the presence of the main eutomer with a concentration as high as 1.0 mg/mL.