The human body is a chiral environment and many drugs are chiral and interact differently depending on the type of enantiomer. Therefore, the interest in analytical and preparative separations of enantiomers has steadily increased over the years. LC is today the most important technique in analytical laboratories worldwide. The key to understand the separation system lies in the adsorption isotherm, which describes the equilibrium distribution of solutes between the mobile and stationary phases. By measuring adsorption isotherms in chiral phase systems, a deeper interpenetration concerning enantioselective and non-selective binding energies and adsorption processes is possible. Furthermore, this data provides the core information needed to optimize preparative chromatographic processes for purification of single enantiomers. However, the measurement of adsorption isotherms is a delicate matter and there are many dangerous pitfalls that may produce erroneous results and even wrong mechanistic conclusions. This review summarizes the most relevant methods and a workflow will be given for avoiding the common pitfalls and obtaining reliable data. Several applications from the literature are also treated to give insight in what information can potentially be obtained from using this methodology.