A high performance liquid chromatographic method for the simultaneous determination of bisindole usambarane alkaloids from the roots of Strychnos usambarensis using a diode-array detector is proposed, The system, consisting of a RP-8 Select B column and a linear gradient elution with acetonitrile and acetate buffer allowed the separation of all the alkaloids in 20 min, Usambarensine was found to be the major tertiary alkaloid present in the roots, In order to demonstrate the possibility of quantifying the bisindole alkaloids in crude roots extracts, the quantitative evaluation of 3',4'-dihydrousambarensine, usambarensine and N(b)-methylusambarensine was carried out. (C) 1998 John Wiley & Sons, Ltd.