The review article covers advances of chiral drugs analysis by high-performance liquid chromatography (HPLC) methods achieved during last 10 years. Emphasis is given to various aspects of influence of biological matrix in pharmacodynamics, pharmacokinetics, HPLC analysis. Discussed is composition of main biological matrices from the point of view of potential interferences to above-mentioned fields of study. Beside typical analytical approaches to chiral recognition in HPLC, sample pretreatment and/or clean-up by conventional extraction procedures, column switching (CSW) techniques using restricted access materials (RAMs), microdialysis (MCD) is discussed. Measurement of unbound drug concentration and discussion of column maintenance and remedy is an additional source of information and field where knowledge on complex properties and interactions of biological matrix is usefully applicable.