Small GTPases are cellular switches that are switched on when bound to GTP and switched off when bound to GDP. Different small GTPase proteins or those with mutations may bind to GTP or GDP with different relative affinities. However, small GTPases generally have very high affinities for guanine nucleotides, rendering it difficult to compare the relative binding affinities for GTP and GDP. Here we developed a method for comparing the relative binding strength of a protein to GTP and GDP using a mant-GDP dissociation assay, whereby the abilities of GTP and GDP to induce the dissociation of bound mant-GDP are compared. This equilibrium type assay is simple, economic, and much faster than obtaining each protein’s affinity for GDP and GTP. The GDP/GTP preference value obtained is useful for comparing the relative GTP/GDP binding preferences of different GTPases or different mutants, even though it is not the real GDP/GTP affinity ratio (but rather an estimation of the ratio).