Inactivation of influenza virus and other potential contaminants like avian adenoviruses coming from embryonated chicken eggs is a critical step in the production of inactivated influenza vaccines. Inactivation must lead to a guaranteed reduction in contaminant titers by at least 4 lg (PFU)/ml. The aim of this study was to identify an optimum cell line for adenovirus propagation and to estimate a reduction in adenovirus titers in vaccine intermediates after inactivation. In a series of experiments, we identified the optimum conditions and the optimum cell line for the propagation of avian adenovirus (strains CELO and Fontes). The most commonly used inactivation methods were analyzed, including inactivation by β-propiolactone and UV light. Viral titers were measured by plaque assays.  After 10 h of inactivation with β-propiolactone, CELO titers fell by 4.12 ± 0.06 lg, whereas Fontes titers, by 4.20 ± 0.19 lg, suggesting that β-propiolactone is an effective inactivating agent. Exposure to UV light led to a reduction in CELO titers by 4.69 ± 0.89 lg and a reduction in Fontes titers by 4.44 ± 1.06 lg after 5 min. N-octyl-β-D-glucopyranoside added at the splitting step reduced CELO titers by 0.93 ± 0.15 lg and Fontes titers by 1.04 ± 0.12 lg, whereas tetradecyltrimethylammonium bromide led to a reduction in CELO and Fontes titers by 1.18 ± 0.17 lg and 1.12 ± 0.38 lg, respectively.