Background: Adenovirus-vectored vaccines played an important role in the global response to SARS-CoV-2. Adenovirus platforms have many advantages including a simple and readily transferred manufacturing process, low cost, and thermostability. Speed of production of an initial Good Manufacturing Practice (GMP)-compliant batch has, however, been viewed as a limitation of adenovirus vectors relative to mRNA platforms. Production of the initial viral starting material and release testing are key rate-limiting steps. Methods: Production of viral starting material from DNA, and release testing in accordance with regulatory expectations, for first-in-human trials of adenovirus-vectored vaccines. Results: We describe experience of these stages in the production of the first GMP batches for multiple adenovirus-vectored candidates and the adaptations made for ChAdOx1 nCoV-19 (the Oxford COVID-19 vaccine) in early 2020. We also report development of a streamlined approach to starting material generation, enabling initial GMP batch availability within c. 60 days of publication of a new pathogen sequence. Using a New World arenavirus vaccine construct as a proof of concept, we demonstrate reproducible execution of this pipeline, maintaining acceptable infectivity and other quality attributes. Conclusions: We discuss opportunities for additional time savings in the future. This work demonstrates suitability of an adenovirus platform to contribute to the “100 Days Mission” for vaccines against “Disease X”.