Gene expression analysis is frequently used to analyze the response to viral infection, and 18S RNA, SHDA and GAPDH represent popular house keeping genes (HKGs) often used to normalize gene expression. Here we describe the first systematic selection and evaluation of suitable HKGs for gene expression analysis in chicken embryo fibroblasts (CEF) infected with NDV adapted to the guidelines from Gorzelniak and Ferguson. Our results indicate that ACTB, HPRT1 and HMBS were valuable and stable HKGs, while 18S RNA, GAPDH and SHDA are considerably regulated during the course of infection and thus precluded for normalization. Normalizing the infection dependent gene IFN-a and the infection independent gene B2M to inappropriate HKGs consequently misleads to significant errors in estimating their regulations. Our study emphasizes that even the most popular HKGs like 18S RNA and GAPDH can lead to divergent and inaccurate data interpretation of significant magnitude if not carefully analyzed for stability before.