Feedlotting cattle is a common strategy to increase growth rate and the quality of the meat from beef cattle. However, the combination of tropical high humidity and climatic temperatures with the carbohydrate and protein rich diet supplied to cattle in this production system can result in deleterious effects on metabolism, heat stress and even heat stroke in extreme cases. Heat stress triggers an inflammatory process which in turn triggers the production of a group of key plasma cytokines: interleukin-1 beta (IL-1β); interleukin-6 (IL-6); interferon gamma (INF-γ) and tumor necrosis factor alpha (TNF-α). These plasma cytokines are found in low concentrations (0.03 to 0.3 pmol/µL) as measured by enzyme-linked immunosorbent assays (ELISA). In this research, we aimed to develop and optimize a mass spectrometry-based assay employing multiple reaction monitoring (MRM-MS) to detect and quantify plasma cytokines at low concentrations. For MRM-MS method development commercially available recombinant cytokines were digested with trypsin and analysed by high resolution MS/MS analysis to select peptides useful for quantification and for MRM design. Samples were chromatographically separated on a Shimadzu Nexera HPLC system (Shimadzu, Australia) using a Phenomenex Kinetex C18 (2.1 x 100 mm) column and a linear gradient of 5-45% acetonitrile over 6 min with a flow rate of 400 μL/min. The eluent from the HPLC was directly coupled to a 6500 QTRAP MS/MS system (AB/Sciex, Foster City, USA) equipped with a TurboV ionization source operated in positive ion mode. Three MRM transitions for each candidate peptide biomarker were selected based on MS response and the collision energy (CE) for each transition was optimised. Using five peptides per cytokine the dynamic range of detection was assessed in aqueous solution. Cytokine extraction will be assessed and optimised using established protocols including solid-phase extraction (SPE) or acetonitrile precipitation. Method development and optimisation experiments have demonstrated that MRM-MS is a sensitive and selective alternative to ELISA capable of identifying and quantifying cytokines at concentrations below 2 nmol/µL.