Recently, it has been shown that nitrile-derivatized phenylalanines possess distinct fluorescent properties depending on the position of the cyano-group within the aromatic ring. These fluorophores have potential as probes for studying protein dynamics due to their sensitivity to local environment. Herein, we demonstrate that 2-cyanophenylalanine (Phe(2CN)) and Phe(4CN) can independently monitor multiple sites during the Ca2+ dependent binding of a skeletal muscle myosin light chain kinase (MLCK) peptide fragment to the protein calmodulin (CaM). These cyano-probes were incorporated at two different positions along the peptide chain and monitored simultaneously via selective excitation of the two chromophores. The peptide was labeled with Phe(4cN) at a residue known to bind to a hydrophobic binding pocket of CaM, while Phe(2cN) was designed to acquire dynamics external to the binding pocket. By selectively exciting each of the chromophores, it was determined that the fluorescence emission of Phe(4cN) located at position 581 of MLCK was quenched in the presence of CaM, while no significant change in Phe(2cN) emission was observed at exposed position 594. The CaM binding affinity (K-d) of the double labeled MLCK peptide was calculated to be approximately 64 nM, which is in agreement with previous measurements. These results indicate that multiple Phe(cN) reporters within the same peptide can simultaneously detect variations in the local environment, and that these fluorophores could be utilized to investigate a wide variety of biological problems.