Fructooligosaccharides (FOS) or oligofructose are unique prebiotics due to their fructosyl units with β (2-1) glycosidic linkage. This natural prebiotic travels intact through the GI tract and provides energy to stimulate the growth of beneficial probiotic microbiota especially Lactobacillus spp. These strains selectively perform anaerobic fermentation of FOS to release diversified favourable SCFAs that has manifold health benefits to human. The aim of this study was to determine the effect of prebiotic FOS on probiotic strains to produce SCFAs. The fermentability of prebiotic microbial FOS (MFOS) in vitro was assessed by comparing with commercial FOS (CFOS) on two probiotic strains Lactobacillus plantarum OUBN2 and Lactobacillus rhamnosus GG ATCC 53103 growth, CFU and type of metabolite synthesized determined by plate count method and gas chromatography, respectively. The results showed that the prebiotic FOS has influenced the growth of Lactobacillus sp. as their counts increased significantly with MFOS than in controls. The effect of 2% MFOS and CFOS on the growth of Lactobacillus plantarum OUBN2 was 5.08 x 10¹⁰ and 3.48 x 10¹⁰ CFU/mL whereas, Lactobacillus rhamnosus GG ATCC 53103 was 4.97 x 10¹⁰ and 4.7 x10¹⁰ CFU/mL, respectively. GC profiles of short-chain fatty acid indicated maximum yield of butyric and propionic acid with concentration in L. plantarum OUBN2 as 44.52 µg/mL and 51.88 µg/mL and L. rhamnosus GG ATCC 53103 was 37.98 µg/mL and 47.08 µg/mL of butyric acid on 2% MFOS and CFOS, respectively. Supplementation of FOS could boost the number of colonic Lactobacillus spp., simultaneously fermenting the FOS to release the health promoting SCFAs namely butyric acid.