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Introduction. The genus Vitex L. counts more than 200 species of shrubs and trees common to subtropical areas of the Earth. Only fruits of Vitex agnus-castus L. are used in official medicine. The aim of our study was to perform the chromatographic analysis of carboxylic acids in the flowering shoots of three Vitex representatives under their cultivation in Ukraine. Materials and Methods. Gas chromatography with mass spectrometry (GC/MS) was applied for the analysis of composition and contents of carboxylic acids in the Vitex agnus-castus L., Vitex negundo L. and Vitex negundo var. cannabifolia (Siebold & Zucc.) Hand.-Mazz. flowering shoots. Results and Discussion. The Vitex agnus-castus raw material contained the most significant amount of short-chained aliphatic acids (2454 mg/kg). Malonic, oxalic, citric, and malic acids noticeably prevailed in all the raw materials. The largest amount of aromatic acids was found in the Vitex negundo var. cannabifolia (3926 mg/kg) flowering shoots. p-Hydroxybenzoic acid dominated among the aromatic acids in the plant raw materials of all species. The most noticeable total content of unsaturated fatty acids was in the Vitex negundo var. cannabifolia (4793 mg/kg). Conclusions. To summarize the contents of valuable carboxylic acids, the obtained results provide the evidence for the development and further pharmacological study of herbal preparations from the flowering shoots of three Vitex species.