Prebiotic is a non-digestible fiber which can pass the digest system to the small intestine which is able to stimulate the growth of beneficial bacteria already resided in the colon. There are hundreds of different types of microorganisms in human gastrointestinal system. It is well known that the colonic microflora have a profound influence on host's health and their populations depend on the diet. One of the predominant culturable probiotic bacterial groups in human colon is bifidobacteria. Although oligosaccharides (OS) are the commonly used prebiotics, most of the currently available oligosaccharides are plant-based: fructo-oligosaccharide (FOS) from chicory roots, xylo-oligosaccharides from hemicellulose, soybean oligosaccharides from soy, isomalto-oligosaccharides from starch, and gentio-oligosaccharides. Among the oligosaccharides, galacto-oligosaccharide (GOS) is the only dairy-based OS. It can be produced from whey lactose, which is the major waste in the cheese industry. Prebiotic oligosaccharides including galacto-oligosaccharides (GOS) have been shown to stimulate the growth of bifidobacteria and other probiotic strains. It is not clear, however, how GOS and other oligosaccharides affect certain intestinal bacterial growth. The objective of this study was to evaluate and compare the effects of GOS from various sources on the growth of selected probiotic strains, including Bifidobacterium breve, B. bifidum, B. longum, Lactobacillus acidophilus and L. plantarum. The tested bacterial strains were cultured in defined media containing various concentrations of GOS as the carbon source in anaerobic bottles and 5-liter fermentors under anaerobic conditions. Three commercialized GOS products and five lab-scale GOS products were tested in order to compare their effects on probiotic bacteria. Cell growth was monitored and used to estimate the specific growth rate and cell biomass produced from the carbon source tested in the fermentation. High performance liquid chromatography (HPLC) was used to analyze the sugar concentrations (glucose, galactose, lactose, 3-OS, 4-OS and 5-OS) and organic acid products (acetic, butyric, and lactic acids) in the fermentation broth. The results showed that the purer the GOS, the higher the specific growth rate for the probiotic bacteria tested. This study confirms the different prebiotic effects of GOS from various sources, including several commercially available oligosaccharides.