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Reduced β-nicotinamide adenine dinucleotide (NADH) is the principal electron donor in glycolytic and oxidative energy metabolism in living cells. Distinctive differences between the excited-state dynamics of free NADH in solution and its intrinsic counterpart in living cells or brain slices are indicated. This chapter investigates the two-photon fluorescence lifetime and rotational anisotropy of NADH, under equilibrium binding conditions with malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), in solution to reconcile these differences. The results indicate an enhancement of the fluorescence lifetime of NADH by a factor of∼ 2 upon MDH enzyme binding, which is consistent with literature values using other biochemical techniques. Furthermore, equilibrated NADH-MDH reveals a complex rotational anisotropy at enzyme concentrations around the K. d value. Analogous measurements on NADH binding with LDH reveal similar trends, albeit different kinetic parameters. The results are compared with lipoamide dehydrogenase where flavin adenine dinucleotide is covalently bound to the enzyme backbone.