Double mutant cycles were constructed using neurotransmitters and synthetic substrates that measure their selective binding to one monoamine oxidase (MAO) enzyme isoform over another as a function of structural change. This work measures a reduction in selectivity for the MAOB isoform of 3 to 9.5 kJ mol-1 upon the addition of hydroxy functional groups to a phenethylamine scaffold. Replacement of hydroxy functional groups on the phenethylamine scaffold by hydrophobic substituents measures an increase in selectivity for MAOB of -1.1 to -6.9 kJ mol-1. The strategies presented here can be applied to the development of competitive reversible inhibitors of MAO enzymes and other targets with structurally related isoforms.