Inducible nitric oxide synthase (iNOS) is induced in different cell types by cytokines and lipopolysaccharide (LPS). Cytokine signal transduction is believed to be mediated primarily through the JAK/STAT pathway. We, therefore, examined the effects of JAKs (Janus Kinase) in the induction of iNOS in macrophages using pharmacological inhibitors by using cell culture, Griess assay, protein assay, and western blotting techniques and also to compare the responses of JAK1and JAK2 inhibitor at different concentrations. The result of this study concludes that JAK inhibitors, i.e., JAK 1 and AG-490 down-regulates multiple signaling pathways such as STAT1-activation, iNOS gene expression and NO production in LPS treated macrophages. JAK2 proved to be an active ingredient and has shown anti-inflammatory effects. Activated JAK2 regulates the phosphorylation of JAK through the activation of PI3K thus plays a pivotal role in LPS-induced iNOS expression.