Escherichia coli (E. coli) continue to be one of the major causes of food poisoning in the world. Different methods have been developed in order to reduce the time for the evaluation of the E. coli vaccines. Infectious bronchitis (IB) is a highly contagious viral disease of poultry causes economic losses. Control of IB virus has been attempted using live attenuated and inactivated vaccines. Due to the continuous emergence of E. coli and infections bronchitis, it was important to find a rapid accurate method of evaluation of the used live attenuated E. coli and IB vaccines. In this study, three assays, namely a conventional identification method including; Specific Pathogen Free eggs (SPF) eggs inoculation for IB vaccine and culture method for E. coli vaccine, conventional polymerase chain reaction (PCR) assay and SYBR Green I Real-Time PCR method were developed and evaluated on 10 fold serial dilutions of each vaccine. A comparative analysis of these three assays was then performed, and the results indicated that the SYBR Green I Real-Time PCR had the highest sensitivity and specificity.