The selection of suitable internal control genes is crucial for proper interpretation of real-time PCR data. Here we outline a strategy to identify housekeeping genes that could serve as suitable internal control for comparative analyses of gene expression data in breast cancer cell lines and tissues obtained by high throughput sequencing and quantitative real-time PCR (qRT-PCR). The strategy proposed includes the large-scale screening of potential candidate reference genes from RNA-seq data as well as their validation by qRT-PCR, and careful examination of reference data from the International Cancer Genome Consortium, The Cancer Genome Atlas and Gene Expression Omnibus repositories. The identified set of reference genes, also called novel housekeeping genes that includes CCSER2, SYMPK, ANKRD17 and PUM1, proved to be less variable and thus potentially more accurate for research and clinical analyses of breast cell lines and tissue samples compared to the traditional housekeeping genes used to this end. These results highlight the importance of a massive evaluation of housekeeping genes for their relevance as internal control for optimized intra- and inter-assay comparison of gene expression. We developed a strategy to identify and evaluate the significance of housekeeping genes as internal control for the intra- and inter-assay comparison of gene expression in breast cancer that could be applied to other tumor types and diseases.