Intracellular flow cytometry is a method of cytokine detection that allows simultaneous detection of intracellular cytokines and cell surface markers. This important method is not extensively used in pigs, in particular due to the inaccessibility of proper methodological protocols modifying comprehensive human protocols. The aim of this study was to find the best procedure for fixation and permeabilization of porcine blood leukocytes and simultaneous cell surface staining. Permeabilization with commercial kits gave better results in most of the chosen parameters compared with combinations of different concentrations of paraformaldehyde and saponin. Among the commercial kits tested, the best results were obtained with the IntraStain kit. Cell surface markers were detected on cells stimulated for cytokine production by antibodies anti-CD14 (clone MIL-2), anti-SWC3, anti-CD4 and anti-CD8 except anti-CD14 (clone Tük4). While anti-CD8 (clone MIL-12) must be used for staining of unfixed cells, the other antibodies recognize fixed and/or permeabilized cells. Moreover, anti-SWC3 and anti-CD14 (clone MIL-2) antibodies can stain cells during the permeabilization step. These modifications of the cell surface staining protocol allow the researcher to speed up the procedure of intracellular cytokine staining or to combine cell surface staining and intracellular cytokine staining. The present study can serve as a particular protocol of intracellular cytokine detection and as a suggestion for optimization of the fixation, permeabilization and cell surface staining procedure in any laboratory.