Macrophages originate from monocytes in the blood, which leave the bloodstream and differentiate into different tissues. Macrophages participate in the detection and phagocytosis of bacteria and damaged cells. In addition, macrophages trigger inflammation by releasing cytokines, which activate vascular cells, promote cytokine adhesion to blood vessels, and migrate to tissues. Differentiated THP-1 cells have been widely used in vitro macrophage models to study the involvement of macrophages in inflammatory responses. The human monocyte line THP-1 can differentiate into macrophages through phorbol 12-tetradecanoate 13-acetate (PMA) and activate them through LPS. Activated THP-1 cells undergo morphological changes and secrete inflammatory cytokines. Monitoring the expression levels of cytokines is an important physiological indicator for inflammatory cell models. We presented the results of multi parameter cell analysis, using phenotype imaging to investigate macrophage formation and low volume ELISA to analyze cytokine secretion, in order to evaluate the effect of pharmacological compounds on inflammatory response. PMA and LPS stimulated THP-1 cells for 48 hours. After PMA and LPS activation of THP-1 cells, IL-8, IL-1b, and TNF-a levels increased. To evaluate anti-inflammatory compounds, cells were treated with kinase inhibitors SB202190 and PDTC, as well as the antibiotic moxifloxacin, before activation. Then, the inhibitory effect of these anti-inflammatory compounds on the inflammatory response was determined by quantifying cytokine secretion levels. The impact of macrophage formation was observed using phenotype imaging. Consistent with the reported mechanism of action, the cytokine expression of compounds SB202190, PDTC, and moxifloxacin decreased in a concentration dependent manner.