Herein we examine the origin of enantioselectivity in the serine protease subtilisin in DMF through the use of molecular dynamics (MD) and free energy perturbation (FEP) simulations. In particular, we are interested in the resolution of a racemic mixture of sec-phenethyl alcohol by a transesterification reaction with the acylating agent vinyl acetate, catalyzed by subtilisin in anhydrous dimethylformamide (DMF). To study the enantioselectivity in this case, we examined the tetrahedral intermediate as a model of the enzyme transition state (as has been done in the past). A critical aspect of this study was the determination of the charge distribution of the two (R and S) tetrahedral intermediates through the use of a combined quantum mechanical/molecular mechanical electrostatic potential fitting methodology. In designing the active site charge model, we found that the R and S tetrahedral intermediates have significantly different charge distributions due to the presence of the stereodifferentiating environment presented by the enzyme. In contrast the charge distribution obtained for models of the tetrahedral intermediate in the gas phase have similar charge distributions, From MD simulations we find that both steric and electrostatic complimentarity plays a role in the enantioselectivity of this enzyme-catalyzed reaction. Through the use of FEP simulations we obtained a free energy difference that is in good accord with experiment, which quantitatively supports the accuracy of our model and suggests that all-atom molecular simulations are capable of providing accurate qualitative and quantitative insights into enzyme catalysis in nonaqueous environments.