Quaternary alkaloids of two Kenyan medicinal plants, Zanthoxylum usambarense and Z. chalybeum, were examined using ion-pair HPLC combined with ion-pair extraction using sodium perchlorate for concentrating quaternary alkaloid fractions. Both plants contained similar alkaloids, but colored protoberberines (jatrorrhizine and palmatine) were found only in Z. chalybeum. A new alkaloid named usambanoline (1) was isolated from the stems of Z. usambarense and characterized. From Z. usambarense, eight known quaternary alkaloids were purified: (+)-tembetarine, (+)-magnoflorine, (+)-N-methylplatydesmine, (-)-oblongine, (-)-cis-N-methylcanadine, nitidine, chelerythrine, and (-)-usambarine, along with (-)-edulinine and the new usambanoline. From Z. chalybeum, similar alkaloids were obtained except (-)-edulinine, plus jatrorrhizine and palmatine. The structure of usambanoline was determined by 2D NMR spectra (1H-1H COSY, 13C-1H COSY, HMBC, NOESY) and SIMS, with a molecular formula of C21H22NO5+ and a structure of 7,8-dimethoxy-3-[6-(2-hydroxyethyl)-1,3-benzodioxolo-5-yl]-2-methylisoquinolinium. Yield analysis showed that the main alkaloids in Z. usambarense were (+)-magnoflorine and (-)-cis-N-methylcanadine, while those in Z. chalybeum were (+)-tembetarine, jatrorrhizine, (-)-cis-N-methylcanadine, and nitidine. The content of nitidine, an antitumor alkaloid, was higher in Z. chalybeum than in Z. usambarense.