OBJECTIVES: Quantification of cytokine is indispensable to elucidate the mechanism of immune regulation at the molecular level, thereby contributing to the diagnosis and treatment of several diseases. Here, we aimed to determine the effect of detection method and storage conditions of specimens on the quantification of cytokine levels. METHODS: We used four vacuum blood collection tubes containing EDTA, heparin, clot activator, gel and compared the changes in the expression levels of 14 cytokines in serum and plasma samples at different temperatures and durations of storage using AimPlex flow cytometry-based immunoassay. In addition, we analyzed the changes in cytokine sensitivity and its clinical relevance after re-establishing cutoff values compared to those set by the manufacturer. RESULTS: The cytokine levels were higher in the plasma of healthy individuals than in their serum. Reliable values for most cytokines were obtained in gel-serum samples, centrifuged within 4 h of collection, and stored at -20 °C for up to 24 h. We measured cytokine levels in 1064 healthy controls using tubes containing separation gel to recalculate their cutoff values and observed changes in cytokine positivity in patients. IL-6 positivity increased from approximately 90% to 95% in patients with sepsis and severe pneumonia. Similarly, IL-8 increased from 48.7% and 41.7% to 79.5% and 63.9% in patients with sepsis and severe pneumonia. CONCLUSIONS: Cytokines are more stable in gel-serum than in plasma or clot activator-serum, and they should be quantified within 4 h or within 24 h if the sample is centrifuged and stored at -20 °C.