Understanding weak interactions in protein–ligand complexes is essential for advancing drug design. Here, we combine experimental and quantum mechanical approaches to study the streptavidin–biotin complex, one of the strongest interacting protein–ligand systems. Using a monomeric streptavidin mutant, we analyze 1H NMR chemical shift perturbations (CSPs) of biotin upon binding, identifying remarkable upfield shifts of up to −3.2 ppm. Quantum chemical calculations attribute these shifts primarily to aromatic ring currents, with additional contributions from charge transfer effects linked to weak interactions. The agreement between experimental and computed chemical shifts validated the X-ray structure as a reliable basis for detailed computational analyses. Energy decomposition analysis reveals that electrostatics dominate the biotin–streptavidin interaction, complemented by significant orbital and dispersion contributions. Notably, weak noncovalent interactions, such as CH···S, CH···π, and CH···HC contacts, driven by London dispersion forces, contribute ∼44% to the complex’s stability.