Computational assessment of the binding affinity of enzyme inhibitors prior to synthesis is an important component of computer-aided drug design (CADD) paradigms. The free energy perturbation (FEP) methodology is the most accurate means of estimating relative binding affinities between two inhibitors. However, due to its complexity and computation-intensive nature, practical applications are restricted to analysis of structurally-related inhibitors. Accordingly, there is a need for methods that enable rapid assessment of a large number of structurally-unrelated molecules in a suitably accurate manner. In this review, the FEP method is compared with molecular mechanics (MM) methods to assess the advantages of each in the estimation of relative binding affinities of inhibitors to an enzyme. Qualitative predictions of relative binding free energies of fructose 1, 6-bisphosphatase inhibitors using MM methods are discussed and compared with the corresponding FEP results. The results indicate that the MM based methods and the FEP method are useful in the qualitative and quantitative assessment of relative binding affinities of enzyme inhibitors, respectively, prior to synthesis.