Traditional analytical methods of determining secondary metabolite levels (e.g., chromatography, spectrophotomety) are based on extraction of the tested tissue and involve its destruction, As a result, it is very difficult to determine such levels in a small localized area. Moreover, because measurements performed in the course of culture necessarily refer to different samples of tissue, levels recorded could reflect variability in the hairy roots themselves, rather than variation as a function of time or morphological location. In this communication we report on a method developed to measure local and overall secondary metabolite levels in a non destructive manner using image analysis and apply it to determine metabolite concentrations in hairy root cultures of Beta vulgaris. To develop the method, after scanning a root under sterile conditions and replacing it in the growth vessel, we converted the image data from red-green-blue to the hue-saturation-intensity coordinate system. Hue and saturation values served respectively for thresholding and determination of pigment level. Root diameter, simulated by thickness of root extract films, was found to be proportional to saturation values, and thus is taken into account when pigment concentration is determined. The data obtained by image analysis were used to determine local and overall accumulation of betacyanin and betaxanthin pigments over time and results were then validated by spectrophotometric analysis. It was concluded that the proposed non destructive method enables accurate determination of changes in local secondary metabolite level at any point in the root, as well as monitoring of changes in overall levels in the entire root system throughout the growth period.