Abstract A systematic study of the separation of a series of tricyclic antidepressant drugs (amitriptyline, nortriptyline, protriptyline, maprotiline, imipramine, chlori-pramine, doxepin-E and doxepin-Z) using reversed phase HPLC with β-cyclodextrin (β-CD) bonded stationary phase is presented. The effect of the organic modifier (methanol) content and pH value of the aqueous phase of the mobile phase on retention was examined and experimental equations of these dependences were derived.The separation is mainly due to the variations of the strength of the CD-solute complexes and this was proved by the good relationship found experimentally between corrected logk' (referred to pure aqueous mobile phase) and the log of complex formation constants. Using the chromatographic response function optimization procedure a mobile phase of 50/50 methanol/buffer pH 6.5 was found as optimum for separations of analytical use. The elution order of the solutes was tried to be correlated to various structure differences.