Chiral separation mechanism determination and absolute configuration assignment are fundamental to the development of chiral stationary phases (CSPs) and the evaluation of both the enantioselective bioactivity and fate of chiral compounds. This work investigated the process of chiral separation and the assignment of the absolute configurations of malathion, malaoxon, and isomalathion using chiral high performance liquid chromatography (HPLC) coupled with chiral detector–binding energy computations. Hydrogen bonding was found to be a very important factor in the chiral separation of isomalathion on Chiralpak AD, although it did not exhibit a significant effect on the chiral separation of malathion and malaoxon on Chiralcel OJ. Based on the sign of a chiral detector, the relationships between the cotton effect, optical dispersion and absolute configuration were established for individual enantiomers of malathion, malaoxon, and isomalathion. The elution orders of the enantiomers of malathion and malaoxon on Chiralcel OJ and the stereoisomers of isomalathion on Chiralpak AD predicted by binding energy computations were found to coincide precisely with those observed in the chiral separation experiments. The result suggests that binding energy computations can be used to assign the absolute configuration of the enantiomers of chiral compounds eluted on CSPs.Highlights► Steric effect dominates chiral separation of malathion and malaoxon. ► Hydrogen bonding is a very important factor in chiral separation of isomalathion. ► The different notations are related for enantiomer of malathion and its metabolites. ► Binding energy computation can be used to predict enantiomeric resolution.