Macrophage activation plays a significant role in homeostasis of organisms. Various internal and external stress factors may affect their function, leading to adverse effects on the body. ‘In vitro macrophage activation techniques provide us with a window to understand the mechanisms of inflammation and response of macrophages to the modulating interventions. Apart from infectious diseases, inflammation is also the major culprit in pathogenesis of many noncommunicable diseases such as arthritis, obesity, metabolic syndrome, diabetes, cancer, cardiovascular disease etc. In vitro macrophage activation allows us to study the role of polarized macrophages in the process of pathogenesis. This emerging technique leads to newer diagnostics, understanding pathophysiological mechanism/s, drug development and management of chronic inflammatory diseases. We, at MRC-KHS, use this technique for screening of medicinal plant-derived phytomolecules for their anti-inflammatory, immunomodulatory and anticancer activities. This review briefly outlines the different experimental models of in vitro macrophage activation and their applications for understanding the pathophysiological mechanisms of underlying chronic inflammation and screening of therapeutic activity of plant-based phytomolecules.