Prebiotics are widely recognized particularly for their role in selectively stimulating the growth and/or activity of beneficial bacteria in the gastrointestinal tract which beneficially affects the host health. Increasing interest has been given to the use of food materials that contain significant amounts of prebiotic components without the necessity of extracting these compounds. In this study, pigeon pea flour was evaluated as a potential prebiotic source. In vitro fermentation of pigeon pea flour by Lactobacillus plantarum (BIOTECH 1223) and Escherichia coli (BIOTECH 1634) was examined based on the changes in cell density, specific growth rate and mean doubling time of the microorganisms, as well as the change in total sugars, resistant starch, dietary fibers (insoluble, soluble and total), pH, titratable acidity of the media. The prebiotic activity score of pigeon pea flour was also determined to measure the extent to which it encourages the selective growth of L. plantarum compared with that of E. coli under the same conditions. Results showed significantly higher growth and metabolic activity of L. plantarum than E. coli in modified medium containing pigeon pea flour. Prebiotic activity score of pigeon pea flour is 0.14 which is not significantly different from the 0.18 prebiotic activity score of commerciallyavailable inulin. Since the prebiotic activity score of pigeon pea flour is comparable to that of inulin, this activity can be extended to other commercially important probiotic organisms and can serve as a rational basis for identifying synbiotics for incorporation into various food products.