Two projects formed the basis of this thesis related to protein-vitamin D₃ (VD₃) binding mechanisms and efficacy. First, a method to characterize the binding of VD₃ to food-related macromolecules that may be used for enrichment of milk was devised. Results suggested that sodium caseinate and hydroxylpropyl-β-cyclodextrin could effectively bind VD₃ and may be used as carriers. Secondly, molecular dynamics simulations were used to determine the conformation ensemble of the β-casein phosphopeptide (β-CPP). Radius of gyration, H-bonding, Ramachandran plot, and secondary structure were ascertained, and showed good agreement with simulations of other disordered pepitides as well as experimental data of β-CPP. Overall, this new binding assay now affords the ability to study interactions between macromolecules and vitamin D₃. As well, by performing simulations of a single casein peptide, important data needed to understand the intramolecular interactions and structure of β-casein (as well as the casein micelle) have be elucidated.