Vitex agnus-castus L. is a medicinal plant which is used in several dosage forms for women hormonal disorders and standardized according to the iridoids or flavonoids content. Aucubin, an iridoid glycoside, considered as a marker in some formulations. In this research, a thin layer chromatographic method with densitometric detection has been developed for quantitative determination of aucubin in chaste tree fruits. Chromatographic separation was performed using silica gel high performance thin-layer chromatography (HPTLC) plates with ethyl acetate-methanol-water 77 : 15 : 8 as mobile phase. Chromatograms were visualized using p-dimethylaminobenzaldehyde as reagent. Aucubin RF-value was about 0.5 and spots were scanned at 580 nm through a mercury lamp. By using this method, the amount of aucubin was found 43.5 mg/100 g of dried plant fruits. The method was validated for selectivity, linearity (r2 = 0.997, 20-100 µg/mL), precision (intra-day < 4.9, inter-day < 7.2) and accuracy measured via determination of recovery (95-98%). The limit of detection and limit of quantization were found 6.6 and 20 µg/mL, respectively. This methodology was found to be precise with respect to the validation parameters. It is simple and convenient and could be applicable to the routine determination of aucubin in different Vitex agnus-castus L. samples.