Abstract In our laboratory, increased demand for cytokine measurements to support both safety and efficacy endpoints across discovery programs has also resulted in cytokine analyses in biological fluids other than routine serum or plasma samples. Recently we have evaluated cytokines in bronchoalveolar lavage (BAL) fluid and CSF to support a mouse respiratory infection and a rat neuroinflammatory model, respectively. Fit-for-purpose validation of these sample types is necessary to confirm cytokine methods are suitable for use, including selection of optimal sample dilutions to reduce or eliminate any matrix effects or interferences. Meso Scale Discovery® (MSD Gaithersburg, MD) methodology was used for measuring cytokines and validation parameters included precision and accuracy of calibration curves (CV% and %Relative Error <20%) to confirm LLOQ and ULOQ for each cytokine method, precision of results for quality control and study samples (CV ≤ 20%) and linearity of response in samples. In development of the mouse respiratory infection model, cytokines IL-1β, IL-6, KC/GRO and MCP-1 were increased in BAL fluid (up to 2892-fold) 20 h post-infection compared to sham and vehicle control groups. Once the model was established, treatment of mice with Compound A demonstrated statistical decreases in these cytokines in BAL fluid compared to an infected control group. Dilutional linearity demonstrated that a minimal required dilution of 1:2 was required for mouse BAL fluids. In support of development of the rat neuroinflammatory model, cytokines IL-1β, IL-6, KC/GRO, MCP-1 and TNF-α were increased (325X, 13X, 28X, 23X and 7.7X, respectively) in CSF compared to plasma at 24 h post treatment. Dilutional linearity evaluations demonstrated that CSF samples can be run neat but a 1:4 dilution was required for plasma samples. In conclusion, fit-for-purpose validation confirmed MSD methods were acceptable for use in supporting these drug discovery models.