Human serum albumin (HSA) plays a fundamental role in the transport of drugs, metabolites, and endogenous ligands. Binding to HSA controls the free, active concentration of a drug, provides a reservoir for a long duration of action, and ultimately affects drug absorption, metabolism, distribution and excretion. The free concentration of a drug can also be affected by interaction with co-administered drugs or by pathological conditions that can modify to a significant extent the binding properties of the carrier, resulting in important clinical impacts for drugs that have a relatively narrow therapeutic index. This manuscript will review the physiological role of albumin in the human body and the pharmacological consequences of drug-albumin binding, and then focus on the structure and the properties of the protein binding sites, as studied by different methodologies. Among these, biochromatography on immobilized albumin has been shown to be a rapid and effective tool for the characterization of albumin binding sites and their enantioselectivity, and for the study of the changes in the binding properties of the protein arising by interaction between different ligands. We will discuss the potential offered by the combined use of circular dichroism on the same protein / drug system in solution, not only for the determination of binding parameters and the detection of displacement phenomena, but also for the identification of conformational features underlying binding stereoselectivity. In particular, the essential role of these methodologies in the study of the enantioselective phenomena occurring in the HSA binding of chiral drugs will be addressed. The effect of reversible or covalent binding of drugs will also be discussed and examples of physiological relevance reported.