Enzyme design is a steadily growing field of computational chemistry, but its successes are limited by the current available knowledge and application of enzyme conformational dynamics. In this work a series of FTIR and 2D IR spectroscopic methods, for observing the conformational dynamics of an enzymatic active site and its surrounding residues, are characterized. The enzyme model system for these studies is the promiscuous ene-reductase from Pyrococcus horikoshii (PhENR) which is capable of binding substrates in multiple orientations. In one method, the spectral lineshape of an aryl-nitrile substrate-analog vibrational label is analyzed using a frequency fluctuation correlation function (FFCF) and compared to the lineshape of a corresponding aryl-azide label. This analysis revealed dynamic and electrostatic active site anisotropy which may influence substrate catalysis. The second method utilizes the intramolecular vibrations of the enzymatic cofactor, flavin mononucleotide (FMN), which is shown to be sensitive to electric field changes associated with substrate binding. The final method places a site-specific nonnatural amino acid containing an azide probe within the enzyme’s hydrophobic core. Additionally, a double-mutant cycle was identified via a common design program, the Rosetta Modeling Suite, and used to analyze the effects of mutation on enzyme dynamics. Altogether, these methods demonstrate the ability of 2D IR spectroscopy to observe enzyme conformational dynamics. Application of these methods to various other enzyme model systems should provide valuable insight for the improvement of future dynamic enzyme design protocols.