Cyclosporine and glucocorticoids are powerful immunosuppressive agents used to treat many inflammatory diseases in dogs. Cyclosporine inhibits calcineurin-dependent pathways of T cell activation and resultant T cell cytokine production, and glucocorticoids directly inhibit genes coding for cytokines. Little work has been done comparing the effects of these agents on T cell cytokine production in dogs. Our study measured T cell interleukin-2 (IL-2) and interferon-gamma (IFN-γ) production using flow cytometry and T cell IL-2 and IFN-γ gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in activated canine T cells incubated with cyclosporine and dexamethasone in vitro. For flow cytometric assays, diluted whole blood was cultured for 7 h in the presence of cyclosporine (10, 100, 500, and 1000 ng/mL) or dexamethasone (10 ng/mL, 100 ng/mL, 1 μg/mL, and 10 μg/mL). For qRT-PCR, whole blood was cultured for 5 h with the same drugs at the same concentrations, and RNA was then extracted from leukocytes. Flow cytometry and qRT-PCR both demonstrated inhibition of IL-2 and IFN-γ that was concentration-dependent in response to cyclosporine, and was more variable for dexamethasone. Quantitative RT-PCR but not flow cytometry documented significant reduction of IL-2 expression after dexamethasone treatment, while both methods showed concentration-dependent suppression of IFN-γ. Quantitative RT-PCR also revealed additional cytokine suppression at higher cyclosporine concentrations, an effect not found using flow cytometry, and may therefore be the preferred method for cytokine determination in dogs. Suppression of IL-2 and IFN-γ in activated T cells may have potential as an indicator of the efficacy of cyclosporine and glucocorticoids in suppressing canine T cell function in vivo, and may therefore be of value for characterizing the immunosuppression induced by these drugs in clinical patients. Copyright © 2019 Elsevier B.V. All rights reserved.