Tuberous sclerosis complex 2 (TSC2), whose gene is frequently mutated in tuberous sclerosis, increases the guanosine triphosphatase (GTPase) activity of the small heterotrimeric GTP-binding protein (G protein) Rheb, thus resulting in the decreased activity of the mammalian target of rapamycin (mTOR), the master regulator of cell growth. Here, we describe the development of a nuclear magnetic resonance (NMR)-based, quantitative, real-time assay to explore the molecular mechanism of the intrinsic and TSC2-catalyzed GTPase activity of Rheb. We confirmed that TSC2 accelerated GTP hydrolysis by Rheb 50-fold through an "asparagine-thumb" mechanism to substitute for the nonfunctional "catalytic" glutamine of Rheb and we determined that catalysis was enthalpy driven. Most, but not all, of the disease-associated GTPase-activating protein (GAP) domain mutants of TSC2 that we examined affected its enzymatic activity. This method can now be applied to study the function and regulation of other GTPases.