The complex process of vaccine product development needs to be tightly controlled and closely monitored to ensure vaccine quality and consistency. Since its inception, PCR has been widely used in all stages of vaccine product development as a tool to assist in the evaluation of vaccine quality, safety and efficacy. In this review, the general principles of conventional and real-time quantitative PCR (Q-PCR) technology and its application in vaccine product development for quantitation of vaccine dose (genome quantitation assay), infectivity (Q-PCR-based potency assay), process residuals, stability, adventitious agents, safety assessment and clinical studies are described. The future outlook and the advantages and disadvantages of this technology are also discussed.