In this study, we developed a macrophage activation monitoring kit based on electrochemical and optical methods for evaluating the activation of macrophages. Macrophages are immune cells, and upon immune activation through external stimuli, they develop pseudopodia-, become irregular in shape, and increase secretion of H2O2. Therefore, using these immune cell properties, we developed a macrophage activation monitoring kit to perform activation testing by measuring macrophage-derived H2O2 in a culture medium. The developed macrophage activation monitoring kit consists of a cell chip and an analysis module, which is designed to be integrated into a cell chip. The developed cell chip contains an analysis section and a cell culture chamber, with a microvalve between them, to allow H2O2 analysis without destroying the cells. In addition, two different analytical methods were introduced to increase sensitivity and efficiency compared to using a single analytical method. For this, the analysis module based on the enzyme-mediated catalytic reaction was developed, and a paper-based analytical device was used for color development reactions, and a carbon paste electrode was used for the electrochemical method. To verify the H2O2 analysis capability of the cell chip, we cultured RAW264.7 cells, a murine macrophage cell line, and activated them using phorbol 12-myristate 13-acetate. As a result, we confirmed that RAW264.7 cells stably adhered and grew on the surface of the cell culture chamber. Furthermore, their activation was confirmed based on cathodic current values, both of which were proportional to RAW264.7 cell concentrations.