The stereochemistry is a prominent feature of living systems and the enantiomers of chiral drugs may often exhibit different pharmacokinetic, pharmacodynamic and toxicological effects in vivo. In fact, they interact with biological macromolecules such as enzymes and receptors that have inherent enantioselectivity. As a result, the drug regulatory agencies currently require studies, not only on the racemate, but also on each enantiomer before conceding approval for marketing of a new chiral drug. Therefore, the pharmaceutical industry has raised its emphasis on the discovery and development of enantiomerically pure compounds, being high-performance liquid chromatography (HPLC) the most widely used analytical tool. The increasing interest in chiral analysis has involved not only the newly developed molecules, but also drugs with long-term use in clinical practice presently re-evaluated as pure enantiomers. Accordingly, in the last few years, many HPLC methods have been developed and validated for the enantioseparation and determination of chiral drugs in several biological samples, to support preclinical and clinical research. Indeed, the availability of an enantioselective analytical method is a critical requirement to assess the chiral discrimination in pharmacokinetic processes, which may explain the differences in biological activity of both enantiomers. This review will focus on the strategies and chiral HPLC techniques developed for the enantiomeric resolution and analysis of chiral drugs. In addition, the impact of the enantioselectivity in pharmacokinetic drug disposition will be also discussed and, when warranted, appropriate pharmacodynamic considerations will be made.