Infectious bronchitis (IB) is a highly contagious viral disease that affects poultry flocks all over the world. Inoculation of specific pathogen free (SPF) embryonated chicken eggs (ECE) is the current protocol for evaluating live infectious bronchitis virus vaccines (either variants or classical virus vaccines). Some variants of IBV vaccines do not cause obvious lesions in the inoculated embryos, which hinder the determination of the titer of those vaccines. The current study was designed to standardize quantitative real-time RT-PCR (RT- qPCR) protocol for quantification of IBV vaccines that will provide accuracy, speed, and ease of use. A total of ten IBV vaccines were used, including both variant and classical virus vaccines. After traditional titration with SPF ECE, a classical IBV vaccine (MA5) was used to develop the standard curve for the RT- qPCR, and the end point titer was expressed as EID50. The optimal harvest time for all IBV vaccinal strains was determined to be 42 hours post inoculation (PI). All the IBV vaccines were titrated in SPF ECEs and the titer of each vaccine was recorded as EID₅₀. Then each of these IBV vaccines was reconstituted in a volume of sterile phosphate buffer saline (PBS) to have 1000 doses/ml, which was divided into two parts. The first was used for direct quantification by RT-qPCR and the second part was inoculated into three SPF ECE via chorioallantoic sac (CAS) of SPF ECE. Using one field dose for each egg. The allantoic fluid was harvested 42 hours post inoculation (hpi), then 200 µL from the pooled allantoic fluid were used for IBV quantification by RT-qPCR. The results indicate that using RT-qPCR for quantification of IBV in live IBV vaccines offers accuracy, speed and ease of use compared to the traditional titration methods.