Stevia rebaudiana (Bertoni) Bertoni is a plant known for its natural sweeteners and potential health benefits. Traditionally used to treat diabetes, its impact on cellular signaling proteins such as PKC is unknown. This study begins with examining a database to analyse publications related to S. rebaudiana. The objective was to identify its experimentally validated phytochemicals and biological targets and to perform network pharmacological analysis of gene-disease associations. The identified targets are subsequently validated using a mouse model for LPS-induced PKC phosphorylation assay on mouse macrophage cells. The effect of HPTLC-standardized S. rebaudiana extract was also evaluated using FACS, ELISA, and fluorescence microscopical analysis. A total of 32 unique phytochemicals were found to interact with three targeted proteins, including 5′-Adenosine Monophosphate (AMP)-activated protein kinase catalytic subunit alpha-2/beta-1/gamma-1, 1,3-beta-D-glucan synthase, and xanthine dehydrogenase/oxidase. Network analysis and molecular modelling also supported targeted lead's binding and inhibition with AMP-activated protein kinase. The LPS significantly enhanced the phosphorylation of PKC in both ex vivo and in vivo assays. The standardised extract, which contains Rebaudioside and Stevioside at 4.82% w/w and 20.12 % w/w, respectively, attenuated the phosphorylation of PKC, suppressed nitric oxide (NO) and reactive oxygen species (ROS). This study found that stevia extract inhibited PKC phosphorylation in macrophages induced by LPS. Additional research is necessary to understand the potential effects of S. rebaudiana in managing PKC-mediated disorders. This includes exploring the mechanisms by which S. rebaudiana may interact with PKC and the possible therapeutic implications for various health conditions associated with PKC dysregulation.