This review attempts to summarize the possibilities offered by the currently most popular separation methods, i.e., high performance liquid chromatography (HPLC) and capillary electrophoresis (CE) for the estimation of dissociation constants of weak protolytes, Separation methods based on the analyte charge are in this respect quite valuable as they can offer the needed information in cases when small amounts of the sample are available, in situations where the compounds are sparingly soluble (or practically insoluble in water) and under favourable circumstances they allow the estimation of close dissociation constants of two (or more) analytes in a single set of experiments, Another advantage of the separation approach of the dissociation constant estimation is the fact that both HPLC and CE can be easily automated. The fact that both HPLC and CE can be run with mobile phases or background electrolytes differing widely in their polarity makes these approaches methods of choice in developing new pharmaceutical products. Adequate attention is paid to data interpretation because while the separation procedures are usually simple, proper interpretation of data obtained is frequently subject in practice to unintended errors.