Difference spectrum is an absorption subtraction method to probe protein structure and dynamics by investigating the environment of specific amino acid residues and the biopolymer backbone in molecular recognition. The difference spectrum offers several advantages over other methods, including low turbidity contributions, high spectral band resolution, low interference and matrix effects, and high signal-to-noise ratio. Additionally, it does not require cosolvent perturbation and can detect oxidized and reduced states in proteins. The method can also investigate conformational transition states involved in electron transfer and proton translocation coupling. Thermodynamic parameters can be accomplished with free or total amounts of ligand or protein, and the data treatment allows investigation for cooperativity, site heterogeneity, and conformational changes in the macromolecule. The chapter describes these approaches to binding hemoglobin with dopamine as a model for molecular interaction by molecular absorption and provides an R-script for the data treatment.