Probiotics have been used as a major alternative approach in the newly hatched chicks to colonize normal micro flora in the intestine. This study involves the characterization of antioxidant potential of probiotic isolates obtained from backyard chicken. Different probiotic isolates namely E. thailandicus, E. hirae, E. faecium, E. fecalis, E. durans, P. acidilactici, L. salivarius were identified by molecular methods using PCR amplification followed by sequencing. Extracellular assays viz DPPH and FRAP assays were performed for assessing the antioxidant potential of these isolates. In vitro assay was also performed in Caco2 and HepG2 cell lines to study the antioxidant changes induced by the probiotic isolates by profiling the antioxidant genes such as SOD (Superoxide dismutase) and GPx (Glutathione peroxidase) with GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) as control by real time PCR. An intricate scoring system was established for evaluating the probiotic isolates for their efficient antioxidant activity. The scoring system revealed that the isolates E. hirae-1, E. faecium-1, E. fecalis-3, P. acidilactici exhibited maximum potential with respect to antioxidant activity.