The use of quantitative real-time reverse transcription-PCR (qRT-PCR) for studying regulation of gene transcription requires an internal template-loading control or a housekeeping gene to guarantee the validity of the data collection, analysis, and interpretation. Analysis of gene transcription in virus-infected animal cells is problematic because virus infection often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes, such as the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and the β-actin gene. It has been demonstrated that the host 28S ribosomal gene can be used as a housekeeping gene in qRT-PCR in virus-infected insect cells. The stability of the human 28S rRNA gene transcription during the infection of HeLa cells with adenovirus has been confirmed, and this method has been extended to the use of the human 28S rRNA gene as a housekeeping gene in adenovirus-infected HeLa cells. Step-by-step instructions are described for use of this control in analysis of gene transcription in both types of virus-infected animal cells.