Cytokine production by leukocytes in response to viral or bacterial pathogens correlates with disease outcomes and therefore carries significant diagnostic value. The goal of the present study was to develop a miniature device for detection of interleukin (IL)-2 and interferon (IFN)-gamma produced by a small population of CD4 and CD8 T-lymphocytes. Microarrays of CD4-, CD8- and cytokine-specific Ab spots were printed onto poly(ethylene glycol) (PEG) hydrogel-coated glass slides and then were enclosed inside a microfluidic device, creating a miniature (3 ul) immunoreaction chamber. Introduction of red blood cell (RBC) depleted whole human blood into the device followed by washing at a pre-defined shear stress resulted in isolation of pure (>95%) CD4+ and CD8+ T-cells on their respective Ab spots. Importantly, immune cells became localized next to anti-IL-2 and -IFN-gamma Ab spots. Mitogenic activation of captured T-cells was followed by immunofluorescent staining (all steps were carried out inside the microdevice) and revealed concentration gradients of IL-2 and IFN-gamma emanating from an array of immune cells. Concentration of IL-2 and IFN-gamma near CD4+ T-cells was estimated to be 30 ng/ml and 20 ng/ml respectively while CD8+ T-cells produced 15 ng/ml of IFN-gamma and no IL-2. The production of cytokines by T-cells was confirmed using intracellular cytokine staining and flow cytometry. This novel immunology tool is envisioned to have applications for HIV diagnosis, vaccine development and monitoring the effectiveness of antiretroviral therapy.