Cytokine and chemokine levels in body fluid provide information of altered conditions in patients. The parallel analysis of multiple factors, such as cytokines, from small sample sizes is an interesting approach for the assessment of in vivo activation signatures and functionality after ex vivo stimulation. One interesting application is for therapy monitoring— such as safety data, pharmacodynamics, evidences for mode-of-action and side effects—which is particularly useful for accompanying early phase clinical trials. There are different platforms for multiplex analyses of ligands available. An assay validation of three different platforms for simultaneous quantification of cytokines (Luminex Bio-Plex® 200, Meso Scale Discovery® and Ella® ) was performed in this study. The comparison of multiplex platforms, which use different ways of achieving parallel measurements of biomarkers, reveal the performance strengths and weaknesses. We showed examples of in-house assessments of intra- and inter-assay variations, determination of the range and recovery of classical immunological serum markers and discussed advantages and disadvantages of these three platforms in relation to the question addressed. All the platforms show low intra-assay variances. The Luminex platform shows a high inter-assay variance for the majority of parameters, whereas the MSD and Ella platforms show low inter-assay variances.