Murine BMDMs are well established as a model primary macrophage population for the study of the inflammatory response. Commonly, LPS is used as a pro-inflammatory stimulus to induce gene expression in BMDMs, to investigate this pro-inflammatory response. Given the efficiency, sensitivity and robustness of RT-qPCR, this technique is commonly employed to measure gene expression levels in the study of the macrophage inflammatory response. The use of RT-qPCR necessitates normalisation of gene expression data to a reference gene, which is stably expressed under the experimental conditions used. In fact, normalisation of GOI data to a reference gene whose expression is regulated by the experimental condition can lead to erroneous results, and therefore incorrect conclusions [17–19]. Thus, the current study aimed to identify the optimal reference genes for normalisation of gene expression data for experiments using BMDMs stimulated with LPS over the course of the inflammatory response, specifically the peak (6 h) and resolution (24 h) phases. The data presented herein demonstrates that the expression levels of “traditional” reference genes may vary significantly in this cell model under pro-inflammatory conditions. Moreover, we demonstrate that the normalisation of GOI data using genes that are regulated by the experimental conditions can have a significant impact on the interpretation of RT-qPCR data and, ultimately, the validity of conclusions drawn.