Intracellular pathogens hijack cell signalling pathways for their own benefit. Here, we describe a novel approach for the comprehensive assessment of host cell signalling response to intracellular pathogens including viruses. The approach involves antibody microarray, gene silencing, and chemical inhibition, and is designed to identify novel targets for anti-infective intervention; components of host cell signalling pathways that are mobilised by intracellular pathogens. First, we studied the signalling response of hepatocytes to hepatitis C virus infection. In addition to confirming the implication of several previously described pathways, we identified a number of novel signalling elements that are either stimulated or suppressed by HCV infection. siRNA silencing and chemical inhibition of MAP4K2, a cell serine/threonine kinase that shows modulation following infection, impairs virus replication, and thus demonstrate that this kinase is a potential therapeutic target for anti-HCV compounds. Subsequently, we deployed this strategy to study the mechanisms whereby the Wolbachia bacterial endosymbiont blocks secondary infections of insect cells by Dengue virus and Zika virus. Antibody microarray revealed that many host cell regulatory proteins were differentially regulated in Wolbachia-infected (vs. non-infected) cells. Gene knockdown and chemical inhibition of the insulin receptor tyrosine kinase, which we showed to be downregulated in Wolbachia-infected cells, revealed that this enzyme is essential for replication of Dengue and Zika viruses in mosquito cells. The results suggest that Wolbachia blocks secondary infection at least partly due to downregulation of insulin receptor. Collectively, the above studies show “proof of principle” that this strategy can be deployed to characterise signalling response of host cells to any intracellular pathogens including viruses, and identify new targets for anti-infective intervention.