Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoESI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time. Copyright © 2005 John Wiley & Sons, Ltd.