Lipopolysaccharide (LPS) has been widely used as a model of immune challenge in pigs as it induces the immediate synthesis of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which trigger the production of the acute phase proteins (APPs) C-reactive protein (CRP), haptoglobin (Hp) and pig-Major Acute Phase Protein (pig-MAP). To measure secreted proteins in porcine plasma, specific and sensitive Enzyme-Linked Immuno Sorbent Assays (ELISAs) are well-suited to perform single parameter analysis, yet this approach is time-consuming and expensive for multi-parameter analyses. During the last decade, multiplex bead-based flow cytometry has been increasingly applied as it offers the opportunity to estimate protein ratios in a small sample volume. Cytometric bead array (CBA) is a flow cytometric application using a diversity of beads with unique fluorescence intensities, covalently coupled to a capture antibody for each protein of interest. Detection antibodies, either directly or indirectly conjugated to a fluorochrome, are added to accomplish the desired sandwich format. The aim of the present study was to develop a CBA 3-plex assay for the major pro-inflammatory cytokines TNF-α, IL-1β and IL-6, and an additional CBA 2-plex assay for the major APPs, CRP and pig-MAP, in porcine plasma. Results were compared to commercial ELISA kits. For the CBA 3-plex assay, the limits of detection (LODs) varied between 0.005 and 0.363ng/mL, the intra- and inter-assay coefficients of variation were <10% and <16%, respectively. For TNF-α, IL-1β, IL-6 and pig-MAP, CBA time-concentration profiles similar to those obtained with commercial ELISAs were observed. In conclusion, the novel validated CBA 3-plex assay provides a fast and economical screening tool for determination of pro-inflammatory cytokine profiles in limited porcine plasma volumes. This tool will be applied to study the immunomodulatory properties of drugs in a porcine LPS inflammation model. This study also demonstrated the applicability of CBA for measurement of APPs in pigs, although a different combination than pig-MAP with CRP is recommended.