RT-qPCR is the method of choice for the accurate detection of low quantities of mRNA due to its higher sensitivity and specificity. Most of the previously published reports about swine cytokine gene expression lack information regarding the validation of the technique, which impedes the potential implementation by new users. This study was focussed on the technical validation of already published RT-qPCR assays for swine proinflammatory (IFN-α, IFN-γ, IL-12p35, IL-12p40 and TNF-α) and immunomodulatory (IL-10 and TGF-β) cytokines, and on defining the best qPCR amplification conditions for simultaneous amplification of the selected cytokines from several porcine tissue samples. The tested RT-qPCR assays here are sensitive (Efficient close to 2, Correlation coefficient higher than 0.95 and a Limit Of Detection below 305-100 mRNA copies), robust (Coefficint of Variation and Factor of Discrimination means were lower than 5 and 3%, respectively) and highly useful for the study of immune swine responses.