Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia coli LPS. Because four of the 29 genes demonstrated poor amplification efficiency due to weak induction of gene expression by LPS, monocytes were stimulated with alternative agents (PMA and Poly I:C) known to induce gene expression in monocytes. These agents, acting through different pathways than LPS, improved the level of gene expression and yielded good amplification efficiency for these genes. PCR efficiency was based on a standard curve for each gene and the calculated efficiency was approximately 100% for all 29 genes. The PCR efficiencies for the majority of the target genes were equivalent to that of the housekeeping gene (18S rRNA) used in all experiments. Dissociation curve analysis and gel electrophoresis revealed a single product for each gene analyzed. To exemplify utilization of the validated primer pairs in studies of inflammatory cell activation, temporal changes in mRNA expression of a subset of genes were monitored in equine monocytes incubated with LPS. The availability of the 29 validated primer pairs reported herein will allow investigators to elucidate the response of horses to a variety of inflammatory stimuli and will further our understanding of disease pathogenesis in horses.