In the last few years, continuous progress in instrumental analytical methodology has been achieved with a substantial increase in the number of new, more specific and more flexible methods for ligand-protein assays. In general, the methods used for drug-protein binding studies can be divided into two main groups: separation methods (enabling the calculation of binding parameters, i.e. the number of binding sites and their respective affinity constants) and non-separation methods (describing predominantly qualitative parameters of the ligand-protein complex). This review will be focussed particularly on recent trends in the development of drug-protein binding methods including stereoselective and non-stereoselective aspects using chromatography, capillary electrophoresis and microdialysis as compared to the "conventional approach" using equilibrium dialysis, ultrafiltration or size exclusion chromatography. The advantages and limitations of various methods will be discussed including a focus on "optimal" experimental strategies taking into account in vitro, ex vivo and/or in vivo studies. Furthermore, the importance of some particular aspects concerning the drug binding to proteins (covalent binding of drugs and metabolites, stereoselective interactions and evaluation of binding data) will be outlined in more detail.